EP1918299A1 - Modulators of cell adhesion molecules and use thereof - Google Patents
Modulators of cell adhesion molecules and use thereof Download PDFInfo
- Publication number
- EP1918299A1 EP1918299A1 EP06123480A EP06123480A EP1918299A1 EP 1918299 A1 EP1918299 A1 EP 1918299A1 EP 06123480 A EP06123480 A EP 06123480A EP 06123480 A EP06123480 A EP 06123480A EP 1918299 A1 EP1918299 A1 EP 1918299A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- necl
- seq
- fusion protein
- amino acids
- antagonist
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 102000016289 Cell Adhesion Molecules Human genes 0.000 title description 7
- 108010067225 Cell Adhesion Molecules Proteins 0.000 title description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 54
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 53
- 238000011282 treatment Methods 0.000 claims abstract description 30
- 230000002776 aggregation Effects 0.000 claims abstract description 19
- 238000004220 aggregation Methods 0.000 claims abstract description 19
- 230000008569 process Effects 0.000 claims abstract description 19
- 230000002314 neuroinflammatory effect Effects 0.000 claims abstract description 14
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 14
- 230000000626 neurodegenerative effect Effects 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 102100024045 Cell adhesion molecule 4 Human genes 0.000 claims description 206
- 102100024646 Cell adhesion molecule 2 Human genes 0.000 claims description 205
- 239000005557 antagonist Substances 0.000 claims description 130
- 102000037865 fusion proteins Human genes 0.000 claims description 94
- 108020001507 fusion proteins Proteins 0.000 claims description 94
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 60
- 150000001413 amino acids Chemical class 0.000 claims description 59
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 56
- 229920001184 polypeptide Polymers 0.000 claims description 44
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 44
- 201000006417 multiple sclerosis Diseases 0.000 claims description 36
- 239000013604 expression vector Substances 0.000 claims description 32
- 150000007523 nucleic acids Chemical class 0.000 claims description 31
- 208000016192 Demyelinating disease Diseases 0.000 claims description 23
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 21
- 108020004707 nucleic acids Proteins 0.000 claims description 17
- 102000039446 nucleic acids Human genes 0.000 claims description 17
- 239000003814 drug Substances 0.000 claims description 16
- 208000015114 central nervous system disease Diseases 0.000 claims description 15
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 14
- 239000003795 chemical substances by application Substances 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 13
- 150000001875 compounds Chemical class 0.000 claims description 8
- 239000012636 effector Substances 0.000 claims description 8
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 claims description 6
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 claims description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 6
- 230000003042 antagnostic effect Effects 0.000 claims description 5
- 239000003085 diluting agent Substances 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 238000003259 recombinant expression Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 abstract description 78
- 102100024649 Cell adhesion molecule 1 Human genes 0.000 abstract description 36
- 238000002360 preparation method Methods 0.000 abstract description 13
- 108010072135 Cell Adhesion Molecule-1 Proteins 0.000 abstract description 12
- 230000003247 decreasing effect Effects 0.000 abstract description 3
- 230000023362 neuron cell-cell adhesion Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 75
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 40
- 239000005090 green fluorescent protein Substances 0.000 description 39
- 201000010099 disease Diseases 0.000 description 36
- 102100024046 Cell adhesion molecule 3 Human genes 0.000 description 30
- 238000003752 polymerase chain reaction Methods 0.000 description 24
- 239000000203 mixture Substances 0.000 description 22
- 239000013612 plasmid Substances 0.000 description 22
- 101000760622 Homo sapiens Cell adhesion molecule 2 Proteins 0.000 description 21
- 102000045218 human CADM2 Human genes 0.000 description 21
- 210000003169 central nervous system Anatomy 0.000 description 20
- 230000003993 interaction Effects 0.000 description 17
- 230000027455 binding Effects 0.000 description 16
- 239000002609 medium Substances 0.000 description 16
- 238000006467 substitution reaction Methods 0.000 description 16
- 241000124008 Mammalia Species 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 12
- 108060003951 Immunoglobulin Proteins 0.000 description 12
- 102000018358 immunoglobulin Human genes 0.000 description 12
- 241000238631 Hexapoda Species 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- 210000004556 brain Anatomy 0.000 description 10
- -1 carrier Substances 0.000 description 10
- 239000003446 ligand Substances 0.000 description 10
- 108091093088 Amplicon Proteins 0.000 description 9
- 206010012305 Demyelination Diseases 0.000 description 9
- 210000003050 axon Anatomy 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 9
- 238000002595 magnetic resonance imaging Methods 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 8
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 8
- 229920002684 Sepharose Polymers 0.000 description 8
- 239000002671 adjuvant Substances 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 208000007118 chronic progressive multiple sclerosis Diseases 0.000 description 8
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 8
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 8
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 7
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 239000011230 binding agent Substances 0.000 description 7
- 239000011347 resin Substances 0.000 description 7
- 229920005989 resin Polymers 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 description 6
- 208000007400 Relapsing-Remitting Multiple Sclerosis Diseases 0.000 description 6
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000004071 biological effect Effects 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 230000000750 progressive effect Effects 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 108090000467 Interferon-beta Proteins 0.000 description 5
- 108010050904 Interferons Proteins 0.000 description 5
- 102000014150 Interferons Human genes 0.000 description 5
- 102000002356 Nectin Human genes 0.000 description 5
- 108060005251 Nectin Proteins 0.000 description 5
- 101100064649 Rattus norvegicus Ehhadh gene Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 230000021164 cell adhesion Effects 0.000 description 5
- 230000003210 demyelinating effect Effects 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000000839 emulsion Substances 0.000 description 5
- 239000012894 fetal calf serum Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 239000002502 liposome Substances 0.000 description 5
- 208000015122 neurodegenerative disease Diseases 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- 206010063401 primary progressive multiple sclerosis Diseases 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- 238000007920 subcutaneous administration Methods 0.000 description 5
- 230000000946 synaptic effect Effects 0.000 description 5
- CXNPLSGKWMLZPZ-GIFSMMMISA-N (2r,3r,6s)-3-[[(3s)-3-amino-5-[carbamimidoyl(methyl)amino]pentanoyl]amino]-6-(4-amino-2-oxopyrimidin-1-yl)-3,6-dihydro-2h-pyran-2-carboxylic acid Chemical compound O1[C@@H](C(O)=O)[C@H](NC(=O)C[C@@H](N)CCN(C)C(N)=N)C=C[C@H]1N1C(=O)N=C(N)C=C1 CXNPLSGKWMLZPZ-GIFSMMMISA-N 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 101000910449 Homo sapiens Cell adhesion molecule 3 Proteins 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 108010005716 Interferon beta-1a Proteins 0.000 description 4
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 4
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- CXNPLSGKWMLZPZ-UHFFFAOYSA-N blasticidin-S Natural products O1C(C(O)=O)C(NC(=O)CC(N)CCN(C)C(N)=N)C=CC1N1C(=O)N=C(N)C=C1 CXNPLSGKWMLZPZ-UHFFFAOYSA-N 0.000 description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 229940124452 immunizing agent Drugs 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229960005027 natalizumab Drugs 0.000 description 4
- 230000004770 neurodegeneration Effects 0.000 description 4
- 239000002687 nonaqueous vehicle Substances 0.000 description 4
- 239000000816 peptidomimetic Substances 0.000 description 4
- 206010036807 progressive multifocal leukoencephalopathy Diseases 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 201000008628 secondary progressive multiple sclerosis Diseases 0.000 description 4
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 239000003826 tablet Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108010072051 Glatiramer Acetate Proteins 0.000 description 3
- 102100026720 Interferon beta Human genes 0.000 description 3
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 3
- 239000012097 Lipofectamine 2000 Substances 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 102000047918 Myelin Basic Human genes 0.000 description 3
- 101710107068 Myelin basic protein Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 238000001261 affinity purification Methods 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 239000008135 aqueous vehicle Substances 0.000 description 3
- 230000003376 axonal effect Effects 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 3
- 230000003959 neuroinflammation Effects 0.000 description 3
- 230000007971 neurological deficit Effects 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 238000004007 reversed phase HPLC Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000600 sorbitol Substances 0.000 description 3
- 235000010356 sorbitol Nutrition 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000000375 suspending agent Substances 0.000 description 3
- 239000012730 sustained-release form Substances 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 208000028698 Cognitive impairment Diseases 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108091006020 Fc-tagged proteins Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 206010018341 Gliosis Diseases 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 101000586618 Homo sapiens Poliovirus receptor Proteins 0.000 description 2
- 108010041012 Integrin alpha4 Proteins 0.000 description 2
- 108010005714 Interferon beta-1b Proteins 0.000 description 2
- 102000003996 Interferon-beta Human genes 0.000 description 2
- 239000012741 Laemmli sample buffer Substances 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 102000006386 Myelin Proteins Human genes 0.000 description 2
- 108010083674 Myelin Proteins Proteins 0.000 description 2
- 102100023302 Myelin-oligodendrocyte glycoprotein Human genes 0.000 description 2
- 102100029740 Poliovirus receptor Human genes 0.000 description 2
- 206010067063 Progressive relapsing multiple sclerosis Diseases 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 101150052859 Slc9a1 gene Proteins 0.000 description 2
- FHEAIOHRHQGZPC-KIWGSFCNSA-N acetic acid;(2s)-2-amino-3-(4-hydroxyphenyl)propanoic acid;(2s)-2-aminopentanedioic acid;(2s)-2-aminopropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound CC(O)=O.C[C@H](N)C(O)=O.NCCCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CCC(O)=O.OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 FHEAIOHRHQGZPC-KIWGSFCNSA-N 0.000 description 2
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 229940003504 avonex Drugs 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000017455 cell-cell adhesion Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 208000010877 cognitive disease Diseases 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000006866 deterioration Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000007884 disintegrant Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 201000002491 encephalomyelitis Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000005713 exacerbation Effects 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 229960003776 glatiramer acetate Drugs 0.000 description 2
- 230000007387 gliosis Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 229940047124 interferons Drugs 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 229960001156 mitoxantrone Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000003068 molecular probe Substances 0.000 description 2
- 210000005012 myelin Anatomy 0.000 description 2
- OVRPUVGBRNDNAS-VIFPVBQESA-N n-[(1s)-1-(2-chloro-6-fluorophenyl)ethyl]-5-cyano-1-methylpyrrole-2-carboxamide Chemical compound N([C@@H](C)C=1C(=CC=CC=1F)Cl)C(=O)C1=CC=C(C#N)N1C OVRPUVGBRNDNAS-VIFPVBQESA-N 0.000 description 2
- 239000002353 niosome Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 229940038850 rebif Drugs 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 210000000434 stratum corneum Anatomy 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 229940079023 tysabri Drugs 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- DIGQNXIGRZPYDK-WKSCXVIASA-N (2R)-6-amino-2-[[2-[[(2S)-2-[[2-[[(2R)-2-[[(2S)-2-[[(2R,3S)-2-[[2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2R)-2-[[2-[[2-[[2-[(2-amino-1-hydroxyethylidene)amino]-3-carboxy-1-hydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxypropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1,5-dihydroxy-5-iminopentylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxybutylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1,3-dihydroxypropylidene]amino]-1-hydroxyethylidene]amino]-1-hydroxy-3-sulfanylpropylidene]amino]-1-hydroxyethylidene]amino]hexanoic acid Chemical compound C[C@@H]([C@@H](C(=N[C@@H](CS)C(=N[C@@H](C)C(=N[C@@H](CO)C(=NCC(=N[C@@H](CCC(=N)O)C(=NC(CS)C(=N[C@H]([C@H](C)O)C(=N[C@H](CS)C(=N[C@H](CO)C(=NCC(=N[C@H](CS)C(=NCC(=N[C@H](CCCCN)C(=O)O)O)O)O)O)O)O)O)O)O)O)O)O)O)N=C([C@H](CS)N=C([C@H](CO)N=C([C@H](CO)N=C([C@H](C)N=C(CN=C([C@H](CO)N=C([C@H](CS)N=C(CN=C(C(CS)N=C(C(CC(=O)O)N=C(CN)O)O)O)O)O)O)O)O)O)O)O)O DIGQNXIGRZPYDK-WKSCXVIASA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- ZORQXIQZAOLNGE-UHFFFAOYSA-N 1,1-difluorocyclohexane Chemical compound FC1(F)CCCCC1 ZORQXIQZAOLNGE-UHFFFAOYSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- XZWXFWBHYRFLEF-FSPLSTOPSA-N Ala-His Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 XZWXFWBHYRFLEF-FSPLSTOPSA-N 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 235000019489 Almond oil Nutrition 0.000 description 1
- 101001007348 Arachis hypogaea Galactose-binding lectin Proteins 0.000 description 1
- RJUHZPRQRQLCFL-IMJSIDKUSA-N Asn-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O RJUHZPRQRQLCFL-IMJSIDKUSA-N 0.000 description 1
- IIFDPDVJAHQFSR-WHFBIAKZSA-N Asn-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O IIFDPDVJAHQFSR-WHFBIAKZSA-N 0.000 description 1
- IQTUDDBANZYMAR-WDSKDSINSA-N Asn-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC(N)=O IQTUDDBANZYMAR-WDSKDSINSA-N 0.000 description 1
- JHFNSBBHKSZXKB-VKHMYHEASA-N Asp-Gly Chemical compound OC(=O)C[C@H](N)C(=O)NCC(O)=O JHFNSBBHKSZXKB-VKHMYHEASA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000024806 Brain atrophy Diseases 0.000 description 1
- 101100148606 Caenorhabditis elegans pst-1 gene Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 206010051290 Central nervous system lesion Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- WYVKPHCYMTWUCW-YUPRTTJUSA-N Cys-Thr Chemical compound C[C@@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)N)O WYVKPHCYMTWUCW-YUPRTTJUSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000004338 Dichlorodifluoromethane Substances 0.000 description 1
- 241000006867 Discosoma Species 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 241001596967 Escherichia coli M15 Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- SSHIXEILTLPAQT-WHFBIAKZSA-N Gln-Asp Chemical compound NC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSHIXEILTLPAQT-WHFBIAKZSA-N 0.000 description 1
- PABVKUJVLNMOJP-WHFBIAKZSA-N Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(O)=O PABVKUJVLNMOJP-WHFBIAKZSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000617808 Homo sapiens Synphilin-1 Proteins 0.000 description 1
- 239000004354 Hydroxyethyl cellulose Substances 0.000 description 1
- 229920000663 Hydroxyethyl cellulose Polymers 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 208000034800 Leukoencephalopathies Diseases 0.000 description 1
- 238000012307 MRI technique Methods 0.000 description 1
- 102100027998 Macrophage metalloelastase Human genes 0.000 description 1
- 101710187853 Macrophage metalloelastase Proteins 0.000 description 1
- 235000019759 Maize starch Nutrition 0.000 description 1
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 1
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 102000003792 Metallothionein Human genes 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 229920000715 Mucilage Polymers 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101100219376 Mus musculus Cadm1 gene Proteins 0.000 description 1
- 241001049988 Mycobacterium tuberculosis H37Ra Species 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 102100035486 Nectin-4 Human genes 0.000 description 1
- 101710043865 Nectin-4 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108010088373 Neurofilament Proteins Proteins 0.000 description 1
- 102000008763 Neurofilament Proteins Human genes 0.000 description 1
- 206010060860 Neurological symptom Diseases 0.000 description 1
- 101100442582 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) spe-1 gene Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- GKZIWHRNKRBEOH-HOTGVXAUSA-N Phe-Phe Chemical compound C([C@H]([NH3+])C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)C1=CC=CC=C1 GKZIWHRNKRBEOH-HOTGVXAUSA-N 0.000 description 1
- FSXRLASFHBWESK-HOTGVXAUSA-N Phe-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 FSXRLASFHBWESK-HOTGVXAUSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 206010040030 Sensory loss Diseases 0.000 description 1
- LZLREEUGSYITMX-JQWIXIFHSA-N Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)N)C(O)=O)=CNC2=C1 LZLREEUGSYITMX-JQWIXIFHSA-N 0.000 description 1
- ILVGMCVCQBJPSH-WDSKDSINSA-N Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CO ILVGMCVCQBJPSH-WDSKDSINSA-N 0.000 description 1
- 208000029033 Spinal Cord disease Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102100021997 Synphilin-1 Human genes 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- VNYDHJARLHNEGA-RYUDHWBXSA-N Tyr-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 VNYDHJARLHNEGA-RYUDHWBXSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000036982 action potential Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 239000008168 almond oil Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- CEGOLXSVJUTHNZ-UHFFFAOYSA-K aluminium tristearate Chemical compound [Al+3].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CEGOLXSVJUTHNZ-UHFFFAOYSA-K 0.000 description 1
- 229940063655 aluminum stearate Drugs 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940021459 betaseron Drugs 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 238000003352 cell adhesion assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 230000033383 cell-cell recognition Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007374 clinical diagnostic method Methods 0.000 description 1
- 238000012761 co-transfection Methods 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 231100000870 cognitive problem Toxicity 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000004624 confocal microscopy Methods 0.000 description 1
- 229940038717 copaxone Drugs 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 235000019404 dichlorodifluoromethane Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 210000003194 forelimb Anatomy 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000008570 general process Effects 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 208000010726 hind limb paralysis Diseases 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000002955 immunomodulating agent Substances 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000016245 inborn errors of metabolism Diseases 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 208000036546 leukodystrophy Diseases 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007108 local immune response Effects 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 210000001259 mesencephalon Anatomy 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- DYKFCLLONBREIL-KVUCHLLUSA-N minocycline Chemical compound C([C@H]1C2)C3=C(N(C)C)C=CC(O)=C3C(=O)C1=C(O)[C@@]1(O)[C@@H]2[C@H](N(C)C)C(O)=C(C(N)=O)C1=O DYKFCLLONBREIL-KVUCHLLUSA-N 0.000 description 1
- 229960004023 minocycline Drugs 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000003007 myelin sheath Anatomy 0.000 description 1
- 230000023105 myelination Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 208000010713 partial hind limb paralysis Diseases 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000011505 plaster Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 239000003380 propellant Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 239000012562 protein A resin Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000013391 scatchard analysis Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000020374 simple syrup Nutrition 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 229940080313 sodium starch Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000001593 sorbitan monooleate Substances 0.000 description 1
- 235000011069 sorbitan monooleate Nutrition 0.000 description 1
- 229940035049 sorbitan monooleate Drugs 0.000 description 1
- VIDRYROWYFWGSY-UHFFFAOYSA-N sotalol hydrochloride Chemical compound Cl.CC(C)NCC(O)C1=CC=C(NS(C)(=O)=O)C=C1 VIDRYROWYFWGSY-UHFFFAOYSA-N 0.000 description 1
- 210000005070 sphincter Anatomy 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 229940032147 starch Drugs 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000002511 suppository base Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- 230000018405 transmission of nerve impulse Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 108010020532 tyrosyl-proline Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/285—Demyelinating diseases; Multipel sclerosis
Definitions
- the present invention relates to new molecules capable of decreasing or altering cell adhesion and especially cell aggregation induced by Necl (Nectin-like)/SynCAM (Synaptic cell adhesion) molecules and their ligands such as other members of the Necl protein family.
- the invention provides proteins useful in the treatment of CNS (central nervous system) disorders, including neuro-inflammatory and neurodegenerative conditions. More particularly, the present invention provides novel proteins and antibodies, DNA encoding thereof, processes for production thereof, pharmaceutical compositions, kits containing thereof and use of these in the preparation of pharmaceutical compositions for the treatment of neuroinflammatory and neurodegenerative conditions.
- Neurodegenerative conditions are characterized by a deterioration or loss of function of nerve cells and other cells of the central nervous system (CNS), which may include injuries or damages at the level of the axons and of synaptic connections.
- CNS central nervous system
- the deterioration of nerve cell function through the progressive destruction of the myelin sheath that protects the nerves and allows uninterrupted transmission of nerve impulses is also called demyelination.
- MS Multiple sclerosis
- This chronic disease is typically characterised by episodes of neurological deficit followed by periods of remission.
- MS is manifested in physical symptoms (relapses and disability progression), Central Nervous System (CNS) inflammation, brain atrophy and cognitive impairment.
- CNS Central Nervous System
- Presenting symptoms include focal sensory deficits, focal weakness, visual problems, imbalance and fatigue. Sexual impairment and sphincter dysfunction may occur.
- MS is now considered to be a multi-phasic disease where periods of clinical quiescence (remissions) occur between exacerbations of the disease. Remissions vary in length (typical frequency of 0.8 - 1.2/year) and may last several years but are infrequently permanent.
- Four courses of the disease are individualized: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP) and progressive relapsing (PR) multiple sclerosis.
- RR relapsing-remitting
- SP secondary progressive
- PP primary progressive
- PR progressive relapsing
- MS is considered to be an autoimmune disorder pathologically characterized by inflammation, demyelination and variable degrees of axonal degeneration and gliosis.
- Demyelination of the axons which can be partial, interferes with the conduction of action potentials along axons, which results in tissue atrophy resulting by the symptoms and neurological deficits and clinical disability experienced by patients.
- MS The diagnosis of MS requires demonstration of dissemination of the disease process in both time and space, and exclusion of other causes. The diagnosis is made clinically with the use of paraclinical tools. The introduction of the McDonald criteria allows a diagnosis of MS to be made after a single clinical event ( Mc Donald et al., 2001, Ann. Neurol., 50:121-127 ). MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction. Advances in cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) have simplified the diagnostic process and facilitated early diagnostic of multiple sclerosis ( Noseworthy et al., 2000, The New England Journal of Medicihe, , 343, 13, 938-952 ). The International Panel on the Diagnosis of MS published revised criteria facilitating the diagnosis of MS and including MRI together with clinical and para-clinical diagnostic methods (Mc Donald et al., 2001, above).
- CSF cerebrospinal fluid
- MRI magnetic resonance imaging
- CAMs Cell adhesion molecules
- Nectins are a group of immunoglobulin (Ig) superfamily proteins that mediate Ca2+-independent cell-cell adhesion through both homophilic as well as heterophilic interactions.
- the nectin family is comprised of nectin -1, -2, -3 and -4.
- Necl-1 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like proteins
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nectin-like 1
- Necl-5 for nect
- SynCAM1 has been found to be expressed in the brain and various organs ( Biederer et al., 2006, Genomics, 87, 139-150 ), while SynCAM3/ Necl-1 is mainly expressed in the nervous system ( Kakunaga et al., 2005, J Cell Sci. 118:1267-77 ).
- Genomic analysis has predicted that SynCAM proteins belong to a set of four proteins: SynCAM 1, 2, 3 and 4 (Biederer et al., 2006; above) for which different nomenclatures have been proposed, in particular nectin-like 1 to 4 (Necl-1 to -4).
- Necl-3 has not been characterized, Necl-4 poorly so ( Fukami et al., 2003, Gene 323:11-18 ), and Necl-5 is only distantly related to Necl-1 to -4.
- T-cell activation occurs first when the receptor of the T-cells sees an antigen in the context of major histocompatibility complex (MHC) class II on the antigen-presenting cell (APC), then the activated T-cells which are initially in the periphery enter the CNS through complex mechanisms involving among other adhesion molecules. After the entry of the activated T cells into the CNS, recruitment of other immune cells occurs, a variety of cytokines are secreted, and ultimately the immune-mediated destruction of the brain parenchyma takes place.
- MHC major histocompatibility complex
- APC antigen-presenting cell
- T-cells In the central nervous system, activated T cells continue to release pro-inflammatory cytokines locally, and the process repeats itself Simultaneously, other immune system cells, including B cells and macrophages, also enter the CNS through the blood-brain barrier, which further enhances the local immune response against myelin. Therefore the interaction of T-cells with adhesion molecules has been shown to be a crucial step in the pathogenesis development of multiple sclerosis.
- Natalizumab targets an adhesion molecule (the ⁇ 4 integrin chain) exemplifies that the interaction of T-cells with adhesion molecules is crucial in the pathogenesis development of multiple sclerosis.
- the present invention is directed towards new molecules capable of decreasing or altering cell adhesion and especially cell-cell contacts induced by Necl-3 and Necl-4 and their ligands, including the other members of the Necl family. More particularly, the present invention provides novel proteins and antibodies, DNA encoding thereof, processes for production thereof, pharmaceutical compositions, kits containing thereof and use of these in the preparation of pharmaceutical compositions for the treatment of CNS disorders such as neuroinflammatory and neurodegenerative conditions.
- a first aspect of the invention provides a polypeptide having at least 80% identity or homology with a sequence of amino acids selected from an amino acid sequence of Necl-3 and an amino acid sequence selected from an amino acid sequence of Necl-4 or fragments thereof.
- a second aspect of the invention relates to nucleic acid molecules encoding a polypeptide as defined above or fusion proteins as described below.
- Such nucleic acids also include vectors containing said molecules, in particular expression vectors.
- a third aspect of the invention resides in a fusion protein having inhibitory activity to a Necl protein such as Necl-1 or Necl-2 or Necl-3 or Necl-4 or a ligand thereof.
- a fourth aspect of the invention relates to antibodies that selectively bind the polypeptides according to the invention, as well as to antibodies that bind Necl-3 (Necl-4) and/or block the binding of Necl-3 (Necl-4) to other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4 and to a use of an antibody according to the invention in an assay.
- a fifth aspect of the invention relates to host cells expressing a polypeptide as defined above, as well as methods of producing such cells.
- a sixth aspect of the invention is a process for preparing a polypeptide or a fusion protein as defined above, typically using recombinant technologies.
- a seventh aspect of the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a polypeptide or a nucleic acid as defined above and a pharmaceutically acceptable carrier or vehicle.
- An eighth aspect of the invention is a use of a Necl-3 antagonist and/or Necl-4 antagonist for the preparation of a medicament for the treatment of CNS disorders.
- a ninth aspect of the invention is a Necl-3 antagonist or a Necl-4 antagonist for use as a medicament.
- a tenth aspect of the invention provides a kit comprising at least one Necl-3 antagonist and/or Necl-4 antagonist according to the invention.
- An eleventh aspect of the invention relates to a method of treating of a disease comprising the administration of a therapeutically effective amount of a Necl-3 antagonist or a Necl-4 antagonist according to the invention in a mammal in need thereof and wherein the disease is a CNS disorders as well as methods of promoting remyelination of nerve cells in a mammal comprising administering to the mammal a Necl-3 and/or Necl-4 antagonist according to the invention.
- a twelfth aspect of the invention is a method of antagonizing the aggregation of Necl-3 and/or of Necl-4 with itself of with at least one protein of the Necl/SynCAM family.
- a thirteenth aspect is a method for in vitro detection of the level of a Necl-3 and of Necl-4 protein in a biological sample.
- a fourteenth aspect is a method for screening for a Necl-3 and/or Necl-4 antagonist.
- neurodegenerative comprises a disease or a state characterized by a CNS degeneration or alteration, especially at the level of the neurons. It comprises neuro-inflammatory and demyelinating states or diseases such as leukoencephalopathies, and leukodystrophies.
- demyelinating is referring to a state or a disease of the CNS comprising the degradation of the myelin around the axons.
- the term demyelinating disease is intended to comprise conditions which comprise a process that demyelinate cells such as multiple sclerosis, progressive multifocal leukoencephalopathy (PML), myelopathies, any neuroinflammatory condition involving autoreactive leukocyte within the CNS, congenital metabolic disorder, a neuropathy with abnormal myelination, drug induced demyelination, radiation induced demyelination, a hereditary demyelinating condition, a prion induced demyelinating condition, encephalitis induced demyelination or a spinal cord injury.
- the condition is multiple sclerosis.
- multiple sclerosis includes relapsing remitting MS (RRMS), Secondary Progressive MS (SPMS), primary progressive MS (PPMS).
- RRMS relapsing remitting MS
- SPMS Secondary Progressive MS
- PPMS primary progressive MS
- Patients suffering from MS can be defined for example as having clinically definite or laboratory-definite MS according to Schumacher or Poser criteria ( Schumacher et al., 1965, Ann. NYAcad. Sci. 1965; 122:552-568 ; Poser et al., 1983, Ann. Neurol. 13(3): 227-31 ).
- efficacy of a treatment according to the invention can be measured based on changes in the course of disease in response to a use according to the invention.
- the efficacy of a treatment of MS can be measured by the frequency of relapses in RRMS and the presence or absence of new lesions in the CNS as detected using methods such as MRI technique ( Miller et al., 1996, Neurology, 47(Suppl 4): S217 ; Evans et al., 1997, Ann. Neurology, 41:125-132 ).
- MRI technique Miller et al., 1996, Neurology, 47(Suppl 4): S217 ; Evans et al., 1997, Ann. Neurology, 41:125-132 .
- Secondary efficacy variables include MRI T 1 enhanced brain lesion volume, MRI T 1 enhanced lesion number, MRI T 2 lesion volume (thought to represent total disease burden, i.e. demyelination, gliosis, inflammation and axon loss), MRI T 1 enhanced hypointense lesion volume (thought to represent primarily demyelination and axon loss), time-to-progression of MS, frequency and severity of exacerbations and time-to-exacerbation, Expanded Disability Status Scale score and Scripps Neurologic Rating Scale (SNRS) score ( Sipe et al., 1984, Neurology, 34, 1368-1372 ).
- SNRS Neurologic Rating Scale
- Degree of disability of MS patients can be for example measured by Kurtzke Expanded Disability Status Scale (EDSS) score ( Kurtzke, 1983, Neurology, 33, 1444-1452 ).
- EDSS Kurtzke Expanded Disability Status Scale
- a decrease in EDSS score corresponds to an improvement in the disease and conversely, an increase in EDSS score corresponds to a worsening of the disease.
- Any other clinical assessment of the disease known is available to the skilled person to measure the efficacy of the treatment according to the invention.
- Relapses involve neurological problems that occur over a short period, typically days but sometimes as short as hours or even minutes. These attacks most often involve motor, sensory, visual or coordination problems early in the disease. Later, bladder, bowel, sexual and cognitive problems may be shown. Sometimes the attack onset occurs over several weeks. Typical MS relapse involves a period of worsening, with development of neurological deficits, then a plateau, in which the patient is not getting any better but also not getting any worse followed by a recovery period. Recovery usually begins within a few weeks.
- treatment and “treating” and the like generally mean obtaining a desired pharmacological and physiological effect.
- the effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease.
- treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions.
- mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses and the like.
- Fc domain encompasses native Fc, including the various subtypes, and Fc variant molecules and sequences as defined below. It includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
- native Fc refers to a molecule or sequence comprising the sequence of a non-antigen binding fragment resulting from the digestion of whole antibody, or by means of recombinant DNA techniques, whether in monomeric or multimeric form.
- the original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins.
- Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e. disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e. g.
- native Fc domains are human IgG1 and IgG4, having respectively the amino acid sequence of SEQ ID NO: 35 and of SEQ ID NO: 36.
- native Fc as used in the context of the invention is generic to the monomeric, dimeric, and multimeric forms.
- Fc variant refers to a molecule or sequence that is modified from a native Fc, for example a molecule or sequence that is humanized from a non-human native Fc.
- a native Fc comprises sites that may be removed or changed because they provide structural features or biological activities that are not required, or alternatively enhanced, for the fusion molecules of the present invention.
- Fc variant comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (i) disulfide bond formation, (ii) incompatibility with a selected host cell (iii) N-terminal heterogeneity upon expression in a selected host cell, (iv) glycosylation, (v) interaction with complement, (vi) binding to an Fc receptor other than a salvage receptor, or (vii) antibody-dependent cellular cytotoxicity (ADCC).
- ADCC antibody-dependent cellular cytotoxicity
- Fc fusion proteins in general refers to molecules comprising at least part of an immunoglobulin Fc domain and at least one peptide. Such fusion proteins may be multimers or dimers or fragment thereof and may be derivatized. Suitable fusion proteins of the invention include a Necl-3 polypeptide e.g. an extracellular domain of Necl-3, a fragment thereof or a mutein thereof, linked to an immunoglobulin Fc region.
- Suitable fusion proteins of the invention include a Necl-4 polypeptide e.g. an extracellular domain of Necl-4, a fragment thereof or a mutein thereof, linked to an immunoglobulin Fc region.
- the extracellular domain, the fragment or the mutein thereof may be fused directly or through linker sequences to the Fc portion of an immunoglobulin.
- the preparation of fusion proteins comprising certain heterologous polypeptides fused to an Fc domain is known in the art WO 91/08298 , WO 96/08570 , WO 93/22332 , WO 04/085478 , WO 01/03737 and WO 02/66514 incorporated by reference in their entirety.
- isolated is used to indicate that the molecule is free of association with other proteins or polypeptides, for example as a purification product of recombinant host cell culture or as a purified extract.
- antibody comprises antibodies binding to Necl-3 or Necl-4 protein or fragment thereof, chimeric antibodies recognizing and/or binding selectively to Necl-3 or Necl-4 protein or fragment thereof, fully human, humanized, genetically engineered or bispecific or multispecific antibodies as well as fragments thereof such as single chain antibodies (scFv) or domain antibodies against Necl-3 and/or Necl-4 protein or fragment thereof and the like.
- Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity.
- the term "selectively" indicates that the antibodies preferentially recognize and/or bind the target polypeptide or epitope, i.e., with a higher affinity than any binding to any other antigen or epitope, i.e. the binding to the target polypeptide can be discriminated from non-specific binding to other antigens.
- the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis ( Scatchard et al., 1949, Ann NY Acad. Sci., 51, 660-672 ).
- monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- antagonists is defined as a molecule that antagonizes completely or partially the activity of biological molecule.
- peptidomimetic is defined as a peptide analog containing non-peptidic structural elements, which peptide is capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic does no longer have classical peptide characteristics such as enzymatically scissile peptide bonds.
- the human Necl-3 amino acid sequence described in SEQ ID NO: 2 has a predicted extracellular domain spanning from amino acids 1 to 367.
- Necl-3 encompasses polypeptides having an amino acid sequence of SEQ ID NO: 2 and fragments thereof such as the corresponding full length extracellularlar domain (aa1-365) of SEQ ID NO: 4 or a fragment thereof (aa169-367) having an amino acid sequence of SEQ ID NO: 15.
- Necl-3 encompasses polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequence of SEQ ID NO: 2, or with the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 15 and which polypeptides are biologically active.
- Necl-3 encompasses Necl-3 secreted peptide where the signal peptide is cleaved. Typically, the signal peptide is cleaved between position 24 and 25 of the SEQ ID NO: 4 as determined by Signal P 3.0 software.
- the nucleotide sequence encoding human Necl-4, isolated as described in Examples 6 and 7, is presented in SEQ ID NO: 5 and the amino acid sequence of human Necl-4 is described in SEQ ID NO: 6.
- the human Necl-4 amino acid sequence described in SEQ ID NO: 6 has a predicted extracellular domain of from amino acids 1 to 323.
- Necl-4 encompasses polypeptides having an amino acid sequence of SEQ ID NO: 6 and fragments thereof such as the full length extracellularlar domain (aal-323) of SEQ ID NO: 8 or a fragment thereof (aa126-322) having an amino acid sequence of SEQ ID NO: 26.
- Necl-4 encompasses polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequence of SEQ ID NO: 6 or with the amino acid sequence of SEQ ID NO: 8 or with the amino acid sequence of SEQ ID NO: 26 and which polypeptides are biologically active.
- Necl-4 encompasses Necl-4 secreted peptide where the signal peptide is cleaved. Typically, the signal peptide is cleaved between position 24 and 25 of the SEQ ID NO: 8 as determined by Signal P 3.0 software.
- Necl-3 variant or a “Necl-4 variant” as referred to herein means a polypeptide substantially homologous to native Necl-3 or native Nelc-4, respectively, but which has an amino acid sequence different from that of native Necl-3 or Necl-4 respectively (human, murine or other mammalian species) because of one or more deletions, insertions or substitutions.
- Substantially homologous means a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the native amino acid sequences, as disclosed above.
- Necl-3 variants or Necl-4 variants may comprise a sequence having at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
- substitutions for one or more amino acids present in the native polypeptide should be made conservatively. Examples of conservative substitutions include substitution of amino acids outside of the active domain(s), and substitution of amino acids that do not alter the secondary and/or tertiary structure of Necl-3 or Necl-4 respectively.
- conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.
- Other such conservative substitutions for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known ( Kyte, et al., 1982, J. Mol. Biol., 157: 105- 131 ).
- Naturally occurring variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the native protein, wherein the native biological property is retained.
- a "conservative amino acid substitution” may involve a substitution of a native amino acid residue with a non native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired.
- Necl-3 or Necl-4 include but is not limited to variants, biologically active fragments, fragments and fragments of variants.
- Necl-3 variants or Necl-4 variants may be used as antagonists of Necl-3 activity or Necl-4 activity or be used to develop Necl-3 antagonists or Necl-4 antagonists, such as antibodies, chimaeric proteins or fusion proteins according to the invention.
- Necl-3 antagonist comprises all antagonists of all suitable forms of Necl-3 described herein that antagonize one or more biological activity of Necl-3 and of Necl-3 variants or fragment thereof.
- the Necl-3 antagonists of the invention are able to antagonize the ability of Necl-3 to participate to homophilic interactions (interactions between Necl-3 proteins) and/or heterophilic interactions (interactions with other protein(s) from the Necl or synCAM family such as Necl-1, Necl-2 and Necl-4, or with other membrane protein ligands).
- Necl-3 antagonist includes but is not limited to: Necl-3 specific antibodies of any sort (polyclonal, monoclonal, antibody fragments, antibody variants), chimaeric proteins, natural or unnatural proteins with Necl-3 antagonizing activities, small molecules, nucleic acid derived polymers (such as DNA and RNA aptamers, PNAs, or LNAs), peptidomimetics, fusion proteins, or gene therapy vectors driving the expression of such Necl-3 antagonists. Further embodiments include as Necl-3 antagonists, soluble Necl-3 fusion proteins such as but not limited to an extracellular domain of Necl-3 or a fragment thereof linked to an Fc domain. Typically, Necl-3 antagonists are able to bind Necl-3 and/or to block the binding of Necl-3 and other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4.
- Necl-4 antagonist comprises all antagonists of all suitable forms of Necl-4 described herein that antagonize one or more biological activity of Necl-4 and of Necl-4 variants.
- the Necl-4 antagonists of the invention are able to antagonize the ability of Necl-4 to participate to homophilic interactions (interactions between Necl-4 proteins) and/or heterophilic interactions (interactions with other protein(s) from the SynCAM family such as Necl-1, Necl-2 and Necl-3, or with other membrane protein ligands).
- the term Necl-4 antagonist includes but is not limited to: Necl-4 specific antibodies, chimeric proteins, small molecules, peptidomimetics and fusion proteins.
- Necl-4 antagonists include as Necl-4 antagonists, soluble Necl-4 fusion proteins such as but not limited to an extracellular domain of Necl-4 or a fragment thereof linked to an Fc domain.
- Necl-4 antagonists are able to bind Necl-3 and/or to block the binding of Necl-4 and other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4.
- Necl-3 antagonist An antibody that is immunoreactive with Necl-3 and/or especially to an extracellular fragment of Necl-3 or a fragment thereof e.g. to an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 15 may be used as a Necl-3 antagonist.
- Necl-4 antagonist An antibody that is immunoreactive with Necl-4 and/or especially to an extracellular fragment of Necl-4 or a fragment thereof e.g. to an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 26 may be used as a Necl-4 antagonist.
- a Necl-3 or a Necl-4 protein, a Necl-3 antagonist or a Necl-4 antagonist, as an isolated, purified or homogeneous protein according to the invention, may be produced by recombinant expression systems as described herein or purified from naturally occurring cells.
- Necl-3, Necl-3 variants or fragments, Necl-3 antagonists, Necl-4, Necl-4 variants or fragments and Necl-4 include prokaryotes, yeast or higher eukaryotic cells.
- Appropriate cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cellular hosts are described for example in Pouwels et al., 1985, Cloning Vectors: A laboratory manual, Elsevier New York .
- Prokaryotes include gram negative and gram positive organism such as E. Coli or Bacilli. Suitable prokaryotic host cells include for example E. Coli BL21 strain. In prokaryotic host cells, such as E. coli, a Necl-3, Necl-3 fragment or variant, Necl-3 antagonist, Necl-4, Necl-4 fragment or variant or Necl-4 antagonist may include a N-terminal methionine residue to facilitate the expression of recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed peptide.
- the invention provides pharmaceutical or therapeutic agents as compositions and methods for treating a patient, preferably a mammalian patient, and most preferably a human patient who is suffering from a medical disorder, and in particular a disorder mediated by Necl-3 and/or Necl-4, such as neuroinflammatory disorders and/or neurodegenerative disorders.
- compositions of the invention can contain one or more Necl-3 antagonist and/or Necl-4 antagonist (including from recombinant and non-recombinant sources) in any form described herein.
- Compositions of this invention may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
- compositions and unit dosages thereof may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
- Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
- Compositions according to the invention are preferably injectable.
- compositions of this invention may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs.
- Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like.
- the compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use.
- Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives.
- Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats.
- Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia.
- Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol.
- Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Further materials as well as processing techniques and the like are set out in Part 5 of Remihgtoh's Pharmaceutical Sciences, 20th Edition, 2000, Marck Publishing Company, Easton, Pennsylvania , which is incorporated herein by reference.
- Solid compositions of this invention may be in the form of tablets or lozenges formulated in a conventional manner.
- tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents.
- Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone.
- Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol.
- Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica.
- Disintegrants include, but are not limited to, potato starch and sodium starch glycollate.
- Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
- Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.
- compositions of this invention may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides.
- Compositions of this invention may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
- Compositions of this invention may also be formulated transdermal formulations comprising aqueous or non-aqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
- compositions of this invention may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion.
- Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents.
- the composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
- compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection.
- the compositions may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
- compositions of this invention may also be formulated as a liposome preparation.
- the liposome preparation can comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell.
- Other suitable formulations can employ niosomes.
- Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
- the compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.
- sustained release materials can also be found in the incorporated materials in Remington's Pharmaceutical Sciences.
- compositions of this invention may be administered in any manner including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal or intranasal administration, or combinations thereof
- Parenteral administration includes, but is not limited to, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intra-thecal, and intra-articular.
- the compositions of this invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion.
- a Necl-3, or a Necl-3 antagonist, or a necl-4 or a Necl-4 antagonist according to the invention are administered intravenously or subcutaneously.
- the dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
- a Necl-3 antagonist and/or a Necl-4 antagonist according to the invention can be administered alone or in combination with a co-agent useful in the treatment of CNS disorders including neuroinflammation and/or neurodegeneration, such as substances useful in the treatment of demyelinating diseases e.g. for example a co-agent selected from IFN-beta, Glatiramer acetate, Corticosteroids, immunusuppressants such as Mitoxantrone, Azathioprine, Cyclophosphamide, Methotrexate, Natalizumab (Tysabri®), metalloproteinases inhibitors, especially MMP-12 and/or MMP-9 inhibitors such as Minocycline.
- a co-agent useful in the treatment of CNS disorders including neuroinflammation and/or neurodegeneration such as substances useful in the treatment of demyelinating diseases e.g. for example a co-agent selected from IFN-beta, Glatiramer acetate, Cor
- the invention encompasses the administration of a Necl-3 antagonist or Necl-4 antagonist of the invention wherein the Necl-3 antagonist or the Necl-4 antagonist is administered to an individual prior to, simultaneously or sequentially with other therapeutic regimens or co-agents useful in the treatment of neuroinflammation and/or neurodegeneration (e.g. multiple drug regimens), in a therapeutically effective amount.
- the Necl-3 antagonist and/or a Necl-4 antagonist according to the invention that are administered simultaneously with said co-agents can be administered in the same or different compositions and in the same or different routes of administration.
- interferon-beta IFN- ⁇
- IFN- ⁇ interferon-beta
- fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
- IFN- ⁇ suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering).
- Rebif® Steono
- Avonex® Biogen
- Betaferon® Schering
- the use of interferons of human origin is also preferred in accordance with the present invention.
- the term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof
- patients according to the invention are patients suffering from CNS disorders such as neuroinflammatory and neurodegenerative disorders or states.
- patients according to the invention are patients suffering from demyelinating states or disorders.
- patients according to the invention are patients suffering from multiple sclerosis.
- nucleic acids encoding Necl-3, Necl-3 antagonists, Necl-4, Necl-4 antagonists and fragments thereof may be used to express recombinant polypeptides for analysis, characterization and therapeutic use.
- Necl-3 antagonists and Necl-4 antagonists according to the invention are useful in the treatment of CNS disorders, including neuroinflammatory and/or neurodegenerative disorders or conditions such as demyelinating diseases or conditions.
- Necl-3 antagonists according to the invention are useful to antagonize one or more biological activity of Necl-3 such as adhesion properties, including Necl-3 induced cell adhesion processes, for example adhesion process between Necl-3 on myelinated axons and cells of the immune system expressing Necl-3 or Necl-3 heterophilic ligands.
- adhesion properties including Necl-3 induced cell adhesion processes, for example adhesion process between Necl-3 on myelinated axons and cells of the immune system expressing Necl-3 or Necl-3 heterophilic ligands.
- Necl-4 antagonists according to the invention are useful to antagonize one or more biological activity of Necl-4 such as adhesion properties, including Necl-4 induced cell adhesion processes, for example adhesion process between Necl-4 on astrocytes and cells of the immune system expressing Necl-4 or Necl-4 heterophilic ligands
- Necl-3 nucleic acid sequences or Necl-4 nucleic acid sequences, or fragments thereof and combinations of fragment thereof may be used as probes or primers.
- Necl-3 or Necl-4 amino acid sequences, variants thereof, fragments thereof and combinations thereof may be used in a process for the preparation of Necl-3 antagonists and/or Necl-4 antagonists according to the invention.
- Necl-3 antibodies and/or Necl-4 antibodies may be used in assays relating for example to the analysis of Necl-3 and/or Necl-4 expression such as western blots, immunohistochemistry, ELISA or FACS assays.
- the beneficial effect includes but is not limited to an attenuation, reduction, decrease or diminishing of the pathological development after onset of the disease.
- Table 2 presents the Sequence identity numbers and associated molecules: Table 2 SEQ ID NO.
- One process for producing Necl-3 antagonists or Necl-4 antagonists comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes a Necl-3 antagonist or a Necl-4 antagonist under conditions sufficient to promote expression of Necl-3 antagonists or Necl-4 antagonists, respectively.
- a Necl-3 antagonist or a Necl-4 antagonist according to the invention is then recovered from culture medium or cell extracts, depending upon the expression system employed. As known to the skilled artisan, procedures for purifying a recombinant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium.
- the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
- a commercially available protein concentration filter for example, an Amicon or Millipore Pellicon ultrafiltration unit.
- the concentrate can be applied to a purification matrix such as a gel filtration medium.
- a purification matrix such as a gel filtration medium.
- an anion exchange and/or an affinity resin can be employed.
- the matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification.
- a cation exchange step can be employed.
- one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media can be employed to further purify Necl-3, Necl-3 variants and Necl-3 antagonists and/or Necl-4, Necl-4 variants and Necl-4 antagonists.
- Recombinant protein produced in bacterial culture can be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps.
- Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
- a desired DNA sequence may be chemically synthesized using techniques known per se.
- DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels.
- Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA fragment into an expression vector, or the fragment may be digested at cleavage sites naturally present therein.
- the well known polymerase chain reaction procedure also may be employed to amplify a DNA sequence encoding a desired protein fragment.
- known mutagenesis techniques may be employed to insert a stop codon at a desired point, e. g. immediately downstream of the codon for the last amino acid of the receptor-binding domain.
- the Necl-3 and/or Necl-4 antagonists are fusion proteins (Fc-Necl-3 and Fc-Necl-4 fusion protein) according to the invention
- they may be prepared according to known processes: for example, the nucleic acid sequence encoding the extracellular domain of Necl-3 or Necl-4 or a fragment thereof can be cloned in an expression vector fused to a nucleic acid sequence encoding the original extracellular domain of Necl-3 or Necl-4 or a fragment thereof signal sequence (or any other appropriate signal/export sequence) at its 5' end, and the nucleic acid sequence encoding the constant region of human immunoglobulin lambda heavy chain IgG (e.g.
- the resulting vector can be used to transform a mammalian (e.g. CHO or HEK293) or an insect (e.g. Schneider S2 cells) host cell line and the clones stably expressing and secreting the recombinant fusion protein having extracellular domain of Necl-3 or Necl-4 or a fragment thereof at the N-terminus and the IgG sequence at the C-terminus can be selected.
- This clone then can be used for scaling up the production and for purifying the recombinant fusion protein from the culture medium.
- the position of the nucleic acid encoding the constant region of human immunoglobulin lambda heavy chain IgG and extracellular domain of Necl-3 or Necl-4 or a fragment thereof can be inversed, and the resulting protein can be expressed and secreted using still the original signal sequence of extracellular domain of Necl-3 or Necl-4 or a fragment thereof, or any other appropriate signal/export sequence.
- Using these technologies it can be also possible to generate heterodimers if two different constructs expressing one Necl-3-Fc or Necl-4-Fc fusion protein and the other a different Fc-based fusion protein (for example another antagonist) are coexpressed in the same host cell ( WO 00/18932 ).
- preparation of the fusion protein according to the invention in insect cells is carried out by the co-transfection of the corresponding insect cell expression vector expressing Fc-Necl-3 extra-cellular domain or Fc-Necl-4 extra-cellular domain (such as illustrated in Examples 6 and 12) along with the plasmid pCoBlast that carries a blasticidin S resistance gene and stable clones are selected with blasticidin S according to methods commonly known in the art.
- high expressers of the fusion proteins are selected.
- the preparation in mammalian cells is carried out by the transfected of the corresponding mammalian cell expression vector expressing Fc-Necl-3 extra-cellular domain or Fc-Necl-4 (such as illustrated in Examples 6 and 12) and stable clones are selected with Zeocin according to methods commonly known in the art. Of these, high expressers of the fusion proteins are selected.
- high expresser clones obtained from one of these methods are expanded and grown (e.g. in a 150 mL flat bottom flask). For instance, S2 insect cells are induced with CuSO 4 (e.g. 0.5 mM). After several days (e.g. 4 days), the conditioned culture medium is loaded onto a Protein A Sepharose column and the Fc-Necl-3 or Fc-Necl-4 fusion protein is recovered according to general methods.
- Necl-3 and/or Necl-4 antagonists are antibodies to Necl-3/Necl-4 according to the invention, they may be prepared according to known processes.
- Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
- an immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
- the immunizing agent may include Necl-3 or Necl-4 or the extracellular fragment or a fragment thereof or a variant as described here above or a fusion protein thereof, or a synthetic peptide encoding a region of Necl-3 or Necl-4. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separated.
- the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in US 4,816,567 .
- the preparation of the polyclonal antibodies according to the invention is performed by using a fragment of the extracellular domain of Necl-3 or Necl-4 (e.g. the Necl-3 (aa169-367) fragment of SEQ ID NO: 15 or Necl-4 (aa126-322) of SEQ ID NO: 26) produced in E. coli to immunize rabbits.
- the same fragment is coupled to NHS activated Sepharose 4 Fast Flow and used for affinity purification of the antibodies from the rabbit sera.
- the antibody-containing fractions are then loaded onto a Protein A Sepharose CL-4B column and the IgGs are eluted.
- the efficacy of the Necl-3/Necl-4 antagonists according to the invention may be assayed in various cell adhesion assays such as described in Biederer et al., 2002, Science. 297:1525-31 or according to the following general process: S2 Drosophila cells are maintained in appropriate medium such as Schneider's Drosophila Medium (Gibco) supplemented with 10% FCS and transfected with an appropriate transfection agent (e.g. Lipofectamine 2000 (Invitrogen), Fugene HD (Roche), or other). After transfection (e.g. about 24 hours) the medium is changed and CuSO 4 is added (e.g. to 0.5 mM) for another period of time (e.g. 24 hours).
- appropriate medium such as Schneider's Drosophila Medium (Gibco) supplemented with 10% FCS and transfected with an appropriate transfection agent (e.g. Lipofectamine 2000 (Invitrogen), Fugene HD (Roche), or other). After
- the cells to be tested are mixed after the medium change, induced with CuSO 4 and gently shaken for several hours to allow for plasmid transcription, protein accumulation and aggregate formation.
- the Necl-3/Necl-4 antagonists and appropriate controls such as unrelated Fc fusion proteins or immunoglobulins are added prior to induction with CuSO 4 and their inhibitory effect on aggregate formation is assessed by microscopy.
- the invention provides an isolated polypeptide having at least 80% (such as at least 85%, at least 90%, at least 95%, at least 98%) identity or homology with a sequence of amino acids selected from the group consisting of:
- the invention provides an isolated polypeptide according to the invention wherein the isolated polypeptide binds to at least one Necl protein or one of its ligand such as Necl-1, Necl-2, Necl-3 or Nec-4 or fragment thereof In a particular embodiment, the isolated polypeptide according to the invention binds to the extracellular fragment or a fragment thereof of at least one Necl protein, such as Necl-1, Necl-2, Necl-3 or Nec-4.
- the invention provides an isolated polypeptide according to the invention comprising an amino acid sequence selected from the group consisting of:
- the invention provides an isolated polypeptide according to the invention comprising an amino acid sequence selected from the group consisting of:
- the invention provides an isolated nucleic acid consisting of a nucleotide sequence encoding a polypeptide having at least 80% (such as at least 85%, at least 90%, at least 95%, at least 98%) identity or homology with a sequence of amino acids selected from the group consisting of:
- the invention provides an isolated nucleic acid consisting of a nucleotide sequence encoding a polypeptide having a sequence of amino acids selected from the group consisting of:
- the invention provides a fusion protein comprising the following sequences:
- the invention provides a fusion protein comprising the following sequences:
- the invention provides a fusion comprising the following sequences:
- the invention provides a fusion protein according to the invention wherein the effector region is a protein with a sequence of amino acid of SEQ ID NO:4.
- the invention provides a fusion protein according to the invention wherein the effector region is a protein with a sequence of amino acid of SEQ ID NO:8.
- the invention provides a fusion protein according to the invention wherein the immunoglobulin constant region is the Fc domain of an IgG.
- the invention provides a fusion protein according to the invention wherein the Fc domain is the Fc domain of an IgG1 or an IgG4.
- the invention provides a fusion protein according to the invention wherein the fusion protein is selected from the following group:
- the invention provides a fusion protein according to the invention wherein the fusion protein is selected from the following group:
- the fusion protein according to the invention of the invention has an inhibitory activity to a Necl protein such as Necl-1 or Necl-2 or Necl-3 or Necl-4 or a ligand thereof.
- the invention provides an isolated DNA sequence that encodes a fusion protein according to the invention.
- the invention provides an isolated antibody or an immunologically active fragment of a monoclonal antibody that binds to at least one Necl protein selected from Necl-3 and Necl-4.
- the invention provides an isolated antibody specifically directed against a polypeptide having a sequence of amino acids selected from the group consisting of:
- the invention provides a use of an antibody according to the invention for the analysis of Necl-3 or Necl-4 expression such as in a western blot, an immunohistochemistry, an ELISA or a FACS assay.
- the invention provides a kit comprising at least one Necl-3 antagonist and/or Necl-4 antagonist according to the invention. More especially, the invention provides a kit comprising at least one Necl-3 antagonist and/or Necl-4 antagonist according to the invention wherein the Necl-3 antagonist and/or Necl-4 antagonist is a fusion protein according to the invention.
- the kit according to the invention may be used for measuring the activity/or the presence of at least one Necl protein such as Necl-1, Necl-2, Necl-3 or Necl-4 or its ligand. In a particular embodiment, the kit is used for measuring the activity/or the presence of Necl-3 and/or Necl-4.
- the invention provides a recombinant expression vector comprising a nucleic acid molecule according to the invention, wherein the vector optionally comprises an expression control sequence, allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide, operably linked to the nucleic acid molecule.
- the invention provides a host cell (e.g. prokaryotic or eukaryotic) transfected or transformed with a recombinant expression vector or a nucleic acid according to the invention.
- a host cell e.g. prokaryotic or eukaryotic
- the invention provides a process for producing cells capable of expressing a polypeptide according to the invention, comprising genetically engineering cells with a vector or a nucleic acid according to the invention
- the invention provides a process for producing a polypeptide according to the invention, comprising transfecting an expression vector according to the invention into a host cell according to the invention under conditions allowing the expression of said polypeptide according to the invention and recovering the said polypeptide.
- the invention provides a process for producing a fusion protein according to the invention, comprising culturing a host cell transformed with an expression vector according to the invention under conditions that promotes expression of said fusion protein and recovering said fusion protein.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising a Necl-3 antagonist or a Necl-4 antagonist according to the invention, and a physiologically acceptable carrier, diluent or excipient.
- the invention provides a pharmaceutical composition wherein the Necl-3 antagonist or the Necl-4 antagonist is selected from a fusion protein according to the invention and a Necl-3 or a Necl-4 specific binding agent of the invention and a physiologically acceptable carrier, diluent or excipient. More especially, the invention provides a pharmaceutical composition comprising a Necl-3 antagonist or a Necl-4 antagonist according to the invention, wherein the Necl-3 antagonist or Necl-4 antagonist is a fusion protein according to the invention, and a physiologically acceptable carrier, diluent or excipient.
- the invention provides a Necl-3 antagonist or a Necl-4 antagonist for use as a medicament.
- the Necl-3 antagonist or the Necl-4 antagonist according to the invention for use as a medicament is selected from a fusion protein according to the invention, an isolated Necl-3 specific binding agent or a Necl-4 specific binding agent according to the invention, and an antibody according to the invention.
- the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases.
- the Necl-3 antagonist or the Necl-4 antagonist according to the invention is selected from a fusion protein according to the invention, an isolated Necl-3 specific binding agent or a Necl-4 specific binding agent according to the invention, and an antibody according to the invention.
- the Necl-3 antagonist or the Necl-4 antagonist according to the invention is a fusion protein according to the invention.
- the Necl-3 antagonist or the Necl-4 antagonist according to the invention is an antibody or an immunologically active fragment of a monoclonal antibody that binds Necl-3 (Necl-4) and/or blocks the binding of Necl-3 (Necl-4) to other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4
- the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases wherein the Necl-3 antagonist or the Necl-4 antagonist is to be administered in combination with a co-agent that useful in the treatment of CNS disorders, including neuroinflammation and/or neurodegeneration such as a co-agent useful in the treatment of demyelination.
- the Necl-3 antagonist or a Necl-4 antagonist according to the invention and the co-agent are used simultaneously or sequentially.
- the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of multiple sclerosis.
- the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases, wherein the Necl-3 antagonist or Necl-4 antagonist is a fusion protein according to the invention.
- the invention provides a method of treating of a disease comprising the administration of a therapeutically effective amount of a Necl-3 antagonist or a Necl-4 antagonist according to the invention in a mammal in need thereof and wherein the disease is a CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases.
- the mammal is human.
- the human suffers from multiple sclerosis.
- the invention relates to methods of promoting remyelination of nerve cells in a mammal comprising administering to the mammal a Necl-3 and/or Necl-4 antagonist according to the invention in a remyelination effective amount.
- the mammal in the methods of the present invention is a human and the human suffers from a condition that demyelinates cells.
- the invention provides a method of antagonizing the aggregation of Necl-3 and/or of Necl-4 with itself of with at least one protein of the Necl/SynCAM family, comprising exposing cells that express Necl-3 or Necl-4, respectively to a Necl-3 antagonist and/or Necl-4 antagonist according to the invention, such that the Necl-3 antagonist or the Necl-4 antagonist blocks the aggregation of Necl-3 or of Necl-4 with itself or with at least one protein of the Necl/SynCAM family.
- the Necl-3 antagonist or Necl-4 antagonist used in this method is a fusion protein according to the invention.
- the invention provides a method for in vitro detection of the level of a Necl-3 and of Necl-4 protein in a biological sample comprising contacting said biological sample with an antibody according to the invention.
- the invention provides a method for screening for a Necl-3 and/or a Necl-4 antagonist, comprising the following steps:
- Amplifications were performed by PCR reaction with Pfu Turbo polymerase (Stratagene) according to the supplier's instructions (1X cloned Pfu buffer, 200 ⁇ M each dNTP, 0.1-0.5 ⁇ M each primer, 2.5 units cloned enzyme, 1-100 ng plasmid template DNA, or 100-250 ng genomic template DNA).
- Mammalian expression vectors pNecl-3-GFP and pNecl3-DsRed2 were constructed as follows:
- the resulting expression vectors were transfected into S2 Drosophila cells that were maintained in Schneider's Drosophila Medium (Gibco) supplemented with 10% FCS.
- cells to be tested were mixed after the medium change, then induced with CuSO 4 and gently shaken several hours to allow for plasmid transcription, protein accumulation and aggregate formation.
- Hela cells were maintained in DMEM 10% FCS and transfected with Fugene (Roche) Fugene was mixed gently before use, then diluted in 100 ⁇ l of medium without serum, and incubated for 5 minutes at room temperature. After 5 minutes of incubation 2 ⁇ g of DNA were added to the diluted Fugene, mixed and incubated for 20 minutes at room temperature. The 100 ⁇ l of complexes were added to each well containing cells and medium.
- the amino acid sequence encoding human Necl-1 and -2 are presented in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
- Necl-1 and Necl-2 protein fused with GFP or with DsRed2 were constructed as follows:
- Example 3 Expression of an extracellular domain of Necl-3 (SEQ ID NO: 14)
- a bacterial expression vector pQE-Necl-3 (aa169-367) was constructed as follows:
- the E. coli strain M15 was transformed with the bacterial expression vector pQE-Necl-3 (aa169-367 ) and used to express the corresponding Necl-3 fragment. Bacteria were grown exponentially, IPTG was added to 1 mM, and the cells were allowed to grow for another 4 h at 37°C. Cells were then harvested and lysed. The cleared supernatants were loaded under denaturing conditions onto Ni 2+ -nitrilotriacetic acid agarose columns (Qiagen, 1 mL resin per 200 mL of bacteria culture), and eluted under standard conditions. The eluted proteins were then extensively dialyzed against PBS and used for immunization.
- the obtained Necl-3 (aa169-367) fragment has an amino acid sequence of SEQ ID NO: 15.
- Example 4 Raising of antibodies against an extracellular domain of Necl-3 (SEQ ID NO: 15)
- Necl-3 (aa169-367) fragment produced in E . coli as described under Example 3 and having the sequence SEQ ID NO: 15 was used to immunize two rabbits according to Harlow et al., 1988, "Antibodies: A Laboratory Manual, " Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 .
- This fragment was coupled to NHS activated Sepharose 4 Fast Flow (Amersham Biosciences) and used for affinity purification of the anti Necl-3 antibodies from the rabbit sera: 10 mg of protein was used for 2 mL of resin. Coupling was performed at 4 °C overnight. After the coupling, non-reacted groups on the medium were blocked with 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 for a one hour. The medium was washed after coupling with 50 mL of two buffers (0.1 M Tris-HCl buffer pH 8-9 and 0.1 M acetate buffer, 0.5 M NaCl). The antibody-containing fractions were then loaded onto a Protein A Sepharose CL-4B column (Amersham Biosciences) and the IgGs were eluted with 0.1 M glycine buffer pH 3.0).
- the antibody specificity was tested using Drosophila S2 cells transfected with either GFP alone or with Necl-1, Necl-2 (obtained as described in Example 2), Necl-3 (as described in Example 1) and Necl-4 (as described in Example 7) fused to GFP at their carboxy-termini where crude S2 lysates were separated by SDS gel electrophoresis and probed with either the anti Necl-3 (obtained as described in Example 4) or an anti GFP antibody (anti-green fluorescent protein, Roche Applied Science) serving as a loading control.
- the anti Necl-3 antibody reveals a single species of the expected size exclusively in cells transfected with Necl-3-GFP which proves that highly specific anti Necl-3 antibodies have been prepared which do not cross-react with other Necl/SynCAM family members, i.e. Necl-1, Necl-2, or Necl-4.
- Necl-3 mRNA expression in eighteen various rat tissues was assessed by real time PCR using a primer pair spanning two exons.
- the primers used for amplifying Necl-3 were SEQ ID NO: 16 (5'-TGACCATGCTCTCATAGG-3') and SEQ ID NO: 17 (5'-TGCCAGATATCGACCAAG-3'), those for GAPDH were SEQ ID NO: 18 (5'-TGATTCTACCCACGGC-3') and SEQ ID NO: 19 (5'-TGATGGGTTTCCCATTGATGA-3').
- RNA purification and cDNA synthesis were prepared as described in ( Ossipow et al., 2004, Mol Cell Neurosci. 2 7:70-83 ).
- Necl-3 protein accumulation was examined by using Necl-3 antibodies obtained as described in Example 4.
- Western blot analysis with extracts from a series of different organs was carried out where rat tissues were heated and sonicated in Laemmli sample buffer. Proteins were separated on a 12% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. These were probed with the appropriate antibodies and revealed by chemiluminescence (ECL, Amersham) (equal amounts of the ECL substrates were mixed and incubated with the blot for 5 min).
- Necl-3 protein was detected in brain structures, but also in kidney, heart and liver. Necl-3 migrates with an apparent mass of approximately 52 kDa (calculated molecular weight of 48 kDa), due to N-linked glycosylation.
- Necl-3 The brain distribution of Necl-3 was investigated by immunohistochemistry and confocal microscopy. 2 month-old rats were anaesthetized, their brains were rapidly excised and immersed for 5 min in isopentane pre-cooled to -20°C. Brains were then embedded in OCT medium (Miles Inc., Elkhart, IN) and cryo-sectioned at 12- ⁇ m.
- Brain sections were exposed for 10 min to -20°C absolute ethanol, rinsed in PBS and incubated for 30 min in PBS containing 2% BSA. Sections were then incubated for 2 h at 37°C in PBS, 0.5% BSA, containing the affinity-purified anti-Necl-3 antibody (diluted 1:100) obtained as described under Example 4 and the appropriate mouse monoclonal antibody. After washes in PBS, Alexa Fluor highly cross-adsorbed secondary antibodies (Molecular Probes) were used to detect the mouse and rabbit primary antibodies (with Alexa 488 and Alexa 594, respectively). The brain sections were then rinsed again in PBS and stained with Hoechst.
- Intense labeling was observed in structures resembling axon bundles in several brain areas including cerebellar white matter and ventral reticular nucleus.
- the axonal localization of Necl-3 was confirmed by a near-perfect overlap with the axonal neurofilament marker.
- Monoclonal antibodies directed against the MBP (Calbiochem) were used in combination with anti Necl-3 antibodies obtained as described under Example 4 to confirm that Necl-3 is found in the axoplasm of myelinated axons and that Necl-3 immunoreactivity is stronger in the myelinated areas.
- S2 cells do not aggregate spontaneously and the ability of Necl-3 to induce their aggregation was assayed.
- S2 cells were transfected separately with plasmids expressing GFP (the insect cell expression vector pMT-GFP was constructed as follows: pEGFP-N1 (BD Biosciences Clonetech) was digested with EcoR1 and Not1, and the resulting insert was ligated into the plasmid pMT (Invitrogen), digested with the same enzymes) or Necl-3-GFP obtained under Example 1 under the control of the drosophila inducible metallothionein promoter. After transfection, the cells were washed free from plasmid, and mixed together.
- GFP the insect cell expression vector pMT-GFP was constructed as follows: pEGFP-N1 (BD Biosciences Clonetech) was digested with EcoR1 and Not1, and the resulting insert was ligated into the plasmid pMT (Invitrogen), digeste
- S2 cells were separately transfected with plasmids coding for Necl-3-GFP (Example 1), Necl-1-DsRed2 (Example 2), and Necl-4-DsRed2 (Example 7). After transfection, they were mixed together in different combinations and induced to express their transfected Necl. After gentle shaking, aggregates formed between Necl-3-GFP and Necl-1-DsRed2 or Necl-4-DsRed2 were observed by fluorescence microscopy. A similar S2 cells aggregation assay using a myc-tagged version of Necl-3 (pNecl-3-myc) along with Necl-2-GFP. Using a rhodamine-conjugated anti myc secondary antibody, aggregates of red and green cells were again observed.
- Necl-3 unambiguously interacts with itself, Necl-1, Necl-2, and Necl-4.
- An Fc-Necl-3 fusion protein between the Fc domain of a human IgG1 of SEQ ID NO: 35 and the extra-cellular domain of Necl-3 (aa 1-365) of SEQ ID NO: 2 was prepared as follows: First, a mammalian expression vector pFc-Necl-3 was constructed by using the plasmid pNecl-3-myc (Example 1) as a template for PCR amplification with the primers 5'-AGGTCTATATAAGC-3' of SEQ ID NO: 12 and 5'-GGGGTACCAGGGCCATTCTGGCC-3 of SEQ ID NO: 13.
- the resulting amplicon was digested with Nhe1 and Kpn1 and ligated into the plasmid pIgplus (R&D Systems, Abingdon, United Kingdom) digested with the same enzymes, resulting in pFc-Necl-3.
- An insect cell expression vector pMT-Fc-Necl-3 was then constructed wherein the pFc-Necl-3 vector obtained above was digested with Nhe1 and Apa1 and the resulting insert was ligated into the plasmid pMT (Invitrogen) digested with Spe1 and Apa1 resulting in pMT-Fc-Necl-3.
- the obtained insect cell expression vector pMT-Fc-Necl-3 was then co-transfected in S2 cells along with the plasmid pCoBlast that carries a blasticidin S resistance gene (Invitrogen). Stable clones were selected with blasticidin S at 25 ⁇ g/mL. Of these, high expressers of the Fc-Necl-3 fusion were selected. One such clone was expanded and grown in a 150 mL flat bottom flask and induced with 0.5 mM CuSO 4 . After 4 days, the conditioned culture medium was loaded onto a Protein A Sepharose CL-4B column (Amersham Biosciences).
- the Fc-Necl-3 fusion protein which was bond to the resin directly from the culture medium was recovered by washing the column (10 volumes of Tris-HCl: 50 mM, pH 7.4) and eluting (0.1 M glycine buffer pH 3.0), and was then characterised by western blotting.
- This fusion protein or a control full-length human IgGs were adsorbed on protein A resin.
- 100 ⁇ l of lysates of mock-transfected and pMT-Necl-2-GFP transfected S2 cells (Example 2) were pre-cleared for 30 min at 4°C with 25 ⁇ l of Protein A Sepharose CL-4B resin and used as the inputs for the following pull-down experiments:
- the lysates were rocked 45 min at 4°C with the Fc-Necl-3 fusion protein immobilized on Protein A Sepharose CL-4B resin. After incubation, the proteins retained on the beads were washed 3 times with 500 ⁇ l RIPA buffer, and boiled in 1X Laemmli sample buffer for western blot analysis. After incubation and washings, the proteins retained on the beads were analyzed by western blot. Control IgG beads were unable to bring down Necl-2-GFP, whereas the Fc-Necl-3 beads efficiently did so.
- Necl-3 is able to make heterophilic interactions with Necl-2, and the fact that human IgGs beads do not interact with Necl-2 confirms that even though the Necl proteins are capable of multiple heterophilic interactions, they retain specificity.
- Necl-4 amplification was performed by PCR reaction with Pfu Turbo polymerase (Stratagene): 1X cloned Pfu buffer, 200 ⁇ M each dNTP, 0.1-0.5 ⁇ M each primer, 2.5 units cloned enzyme, 1-100 ng plasmid template DNA, or 100-250 ng genomic template DNA).
- Insect expression vectors pMT-Necl-4-GFP and pMT-Necl-4-DsRed2 were constructed as follows:
- a mammalian expression vector pNecl-4-GFP was constructed as follows:
- a bacterial expression vector pQE-Necl-4 (aa126-322) was constructed as follows:
- the insect vectors were transfected into S2 Drosophila cells under the same conditions as described for the transfection conditions described in Example 1.
- Example 8 Expression of Necl-4 an extracellular domain (SEQ ID NO: 26)
- a M15 E. coli strain was transformed with the bacterial expression vector pQE-Necl-4 (aa126-322) obtained above and used to express the corresponding Necl-4 fragment.
- the bacteria were grown exponentially, 1 mM IPTG was added, and the cells were allowed to grow for another 4 h at 37°C. Cells were then harvested and lysed. The cleared supernatants were loaded under denaturing conditions onto Ni 2+ -nitrilotriacetic acid agarose columns (Qiagen), i.e. 1 mL resin per 200 mL of bacteria culture, and eluted under standard conditions. The eluted proteins were then extensively dialyzed against PBS and used for immunization.
- the obtained Necl-4 (aa126-322) fragment has an amino sequence of SEQ ID NO: 26.
- Example 9 Raising of antibodies against an extracellular domain of Necl-4 (SEQ ID NO:26)
- Necl-4 (aa126-322) fragment produced in E. coli as described under Example 7 and having the amino acid sequence of SEQ ID NO: 26 was used by to immunize two rabbits on the same manner as described under Example 4.
- Necl-4 expression was examined in various tissues by real-time PCR in the way as described under Example 5 and wherein the primers used for amplifying Necl-4 were 5'-GAGGGCGCTCTACGTACTTG-3' of SEQ ID NO: 27 and 5'-AACCGACGTCTGAGCCTCTA-3' of SEQ ID NO: 28, those for GAPDH were of SEQ ID NO: 18 and SEQ ID NO: 19.
- the Necl-4 expression was shown to be the highest in brain and spinal cord.
- Necl-4 to form homophilic interaction was demonstrated as described under Example 6 wherein the S2 cells were transfected with the plasmid expressing Necl-4-GFP obtained under Example 7.
- Necl-4 The ability of Necl-4 to form heterophilic interaction was demonstrated as described under Example 6 wherein the S2 cells were separately transfected with the plasmid expressing Necl-4-GFP obtained under Example 7, the plasmid coding for Necl-3-GFP obtained under Example 1, the plasmids coding for Necl-4-DsRed2 obtained under Example 7, Necl-1-GFP and Necl-2-GFP obtained under Example 2.
- Necl-4 showed to unambiguously interact with itself, Necl-1, Necl-2, and Necl-3.
- the following fusion proteins for modulating the Necl-3 and/or Necl-4 induced cell aggregation are prepared as described in Example 6.
- the fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- the fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- the fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the mouse Fc domain of an IgG2a of SEQ ID NO: 37;
- the fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the mouse Fc domain of an IgG3 of SEQ ID NO: 38;
- the fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- the fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- the fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the mouse Fc domain of an IgG2a of SEQ ID NO: 37;
- the fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the mouse Fc domain of an IgG3 of SEQ ID NO: 38.
- Necl-3 antagonists or Necl-4 antagonists and in particular of fusion proteins according to the invention in the treatment of CNS disorders may be assayed in different models such as Experimental Autoimmune Encephalomyelitis (EAE) model such as described in Loo et al., 2006, Nature Immunology 7(9): 954-961 .
- EAE Experimental Autoimmune Encephalomyelitis
- Necl-3 antagonists or Necl-4 antagonists according to the invention may be subjected to i.v. injection with various doses and dosing regimens in mice primed to develop (EAE).
- MOG peptide SEQ ID NO: 41 (Sigma-Aldrich Co.) (Sigma-Aldrich Co.) is injected into mice of 10-15 weeks of age by subcutaneous injection of 200 ⁇ g peptide in 200 ⁇ l sterile PBS emulsified with an equal volume of complete Freund's adjuvant (Sigma) containing 5 mg/mL of Mycobacterium tuberculosis H37Ra (BD).
- Necl-3 antagonists or Necl-4 antagonists according to the invention are assigned with scores on a scale of 0-6, with 0.5 points for immediate clinical findings, as follows: 0, normal; 1, weakness of the tail; 2, complete loss of tail tonicity; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, forelimb paralysis or moribund; and 6, death.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Neurosurgery (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Toxicology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Public Health (AREA)
- Neurology (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Psychiatry (AREA)
- Hospice & Palliative Care (AREA)
- Cell Biology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention is related to new molecules capable of decreasing or altering neuronal cell adhesion and especially cell aggregation induced by Necl/SynCAM molecules. In particular, the invention provides proteins useful in the treatment of neuro-inflammatory and neurodegenerative conditions. More particularly, the present invention provides novel proteins and antibodies, DNA encoding thereof, processes for production thereof, pharmaceutical compositions, kits containing thereof and the use of these in the preparation of pharmaceutical compositions for the treatment of neuroinflammatory and neurodegenerative conditions.
Description
- The present invention relates to new molecules capable of decreasing or altering cell adhesion and especially cell aggregation induced by Necl (Nectin-like)/SynCAM (Synaptic cell adhesion) molecules and their ligands such as other members of the Necl protein family. In particular, the invention provides proteins useful in the treatment of CNS (central nervous system) disorders, including neuro-inflammatory and neurodegenerative conditions. More particularly, the present invention provides novel proteins and antibodies, DNA encoding thereof, processes for production thereof, pharmaceutical compositions, kits containing thereof and use of these in the preparation of pharmaceutical compositions for the treatment of neuroinflammatory and neurodegenerative conditions.
- Neurodegenerative conditions are characterized by a deterioration or loss of function of nerve cells and other cells of the central nervous system (CNS), which may include injuries or damages at the level of the axons and of synaptic connections. The deterioration of nerve cell function through the progressive destruction of the myelin sheath that protects the nerves and allows uninterrupted transmission of nerve impulses is also called demyelination.
- Multiple sclerosis (MS) is the most known chronic inflammatory demyelinating disease of the central nervous system in humans. The onset of the disease typically occurs during
ages 20 to 40. Women are affected approximately twice as often as men. It is estimated to affect about 2.5 million people worldwide and its economic impact is considerable. - This chronic disease is typically characterised by episodes of neurological deficit followed by periods of remission. MS is manifested in physical symptoms (relapses and disability progression), Central Nervous System (CNS) inflammation, brain atrophy and cognitive impairment. Presenting symptoms include focal sensory deficits, focal weakness, visual problems, imbalance and fatigue. Sexual impairment and sphincter dysfunction may occur.
- Approximately half of the patients with MS may experience cognitive impairment or depression. MS is now considered to be a multi-phasic disease where periods of clinical quiescence (remissions) occur between exacerbations of the disease. Remissions vary in length (typical frequency of 0.8 - 1.2/year) and may last several years but are infrequently permanent. Four courses of the disease are individualized: relapsing-remitting (RR), secondary progressive (SP), primary progressive (PP) and progressive relapsing (PR) multiple sclerosis. A total of 85 - 90% of patients experience a relapsing/remitting course, which over time becomes secondary progressive in the majority of patients; 10 - 15% have a progressive course from the onset (primary progressive MS). Over time, the relapse frequency declines.
- MS is considered to be an autoimmune disorder pathologically characterized by inflammation, demyelination and variable degrees of axonal degeneration and gliosis. Demyelination of the axons, which can be partial, interferes with the conduction of action potentials along axons, which results in tissue atrophy resulting by the symptoms and neurological deficits and clinical disability experienced by patients.
- The diagnosis of MS requires demonstration of dissemination of the disease process in both time and space, and exclusion of other causes. The diagnosis is made clinically with the use of paraclinical tools. The introduction of the McDonald criteria allows a diagnosis of MS to be made after a single clinical event ( Mc Donald et al., 2001, Ann. Neurol., 50:121-127 ). MS onset is defined by the occurrence of the first neurological symptoms of CNS dysfunction. Advances in cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) have simplified the diagnostic process and facilitated early diagnostic of multiple sclerosis ( Noseworthy et al., 2000, The New England Journal of Medicihe, , 343, 13, 938-952 ). The International Panel on the Diagnosis of MS published revised criteria facilitating the diagnosis of MS and including MRI together with clinical and para-clinical diagnostic methods (Mc Donald et al., 2001, above).
- Cell adhesion molecules (CAMs) are plasma membrane proteins specialized in cell-cell recognition and adhesion. Nectins are a group of immunoglobulin (Ig) superfamily proteins that mediate Ca2+-independent cell-cell adhesion through both homophilic as well as heterophilic interactions. The nectin family is comprised of nectin -1, -2, -3 and -4. In addition, five nectin-like proteins, also called SynCAMs (synaptic CAM), have been identified and named Necl-1 (for nectin-like 1) through Necl-5 ( Ikeda et al., 2003, The Journal of Biological Chemistry, 278, 30, 28167-28172 ; Fuchs et al., 2006, Seminars in Cancer Biology, 16, 359-366 ). Two SynCAMs (SynCAM1, alternatively named Necl-2 and SynCAM3, alternatively named Necl-1) have been shown to serve as synaptic adhesion molecule in the CNS. SynCAM1 has been found to be expressed in the brain and various organs ( Biederer et al., 2006, Genomics, 87, 139-150 ), while SynCAM3/ Necl-1 is mainly expressed in the nervous system ( Kakunaga et al., 2005, J Cell Sci. 118:1267-77 ). Genomic analysis has predicted that SynCAM proteins belong to a set of four proteins: SynCAM 1, 2, 3 and 4 (Biederer et al., 2006; above) for which different nomenclatures have been proposed, in particular nectin-like 1 to 4 (Necl-1 to -4). Necl-3 has not been characterized, Necl-4 poorly so ( Fukami et al., 2003, Gene 323:11-18 ), and Necl-5 is only distantly related to Necl-1 to -4.
- Multiple sclerosis pathogenesis is described as a succession of immunological events. Although the exact course is not fully understood yet, it is believed that the following events are essential. T-cell activation occurs first when the receptor of the T-cells sees an antigen in the context of major histocompatibility complex (MHC) class II on the antigen-presenting cell (APC), then the activated T-cells which are initially in the periphery enter the CNS through complex mechanisms involving among other adhesion molecules. After the entry of the activated T cells into the CNS, recruitment of other immune cells occurs, a variety of cytokines are secreted, and ultimately the immune-mediated destruction of the brain parenchyma takes place. In the central nervous system, activated T cells continue to release pro-inflammatory cytokines locally, and the process repeats itself Simultaneously, other immune system cells, including B cells and macrophages, also enter the CNS through the blood-brain barrier, which further enhances the local immune response against myelin. Therefore the interaction of T-cells with adhesion molecules has been shown to be a crucial step in the pathogenesis development of multiple sclerosis.
- Current medications for MS which are disease modifying treatments, i.e. modifying the course of MS, modulate or suppress the immune system. There are four immunomodulating agents approved by the Food and Drug Administration (FDA) for RRMS: three beta interferons (Betaseron®, Berlex; Avonex®, Biogen; Rebif®, Serono) and Glatiramer Acetate (Copaxone®, Amgen). There is also one FDA approved immunosuppressing drug for worsening MS, Mitoxantrone (Novantrone®, Amgen) and a humanized monoclonal antibody directed against the human α4 integrin chain, Natalizumab (Tysabri®, Biogen and Elan). Currently, the most effective MS treatment is Natalizumab but its clinical use is somewhat limited by its potential side effects. The fact that Natalizumab targets an adhesion molecule (the α4 integrin chain) exemplifies that the interaction of T-cells with adhesion molecules is crucial in the pathogenesis development of multiple sclerosis.
- Due to the severity of the disease, the limited efficacy of the known treatments and the chronic character of the neurodegenerative diseases, it would be highly desirable to have a new method for treating neurodegenerative disorders, such as multiple sclerosis.
- The present invention is directed towards new molecules capable of decreasing or altering cell adhesion and especially cell-cell contacts induced by Necl-3 and Necl-4 and their ligands, including the other members of the Necl family. More particularly, the present invention provides novel proteins and antibodies, DNA encoding thereof, processes for production thereof, pharmaceutical compositions, kits containing thereof and use of these in the preparation of pharmaceutical compositions for the treatment of CNS disorders such as neuroinflammatory and neurodegenerative conditions.
- A first aspect of the invention provides a polypeptide having at least 80% identity or homology with a sequence of amino acids selected from an amino acid sequence of Necl-3 and an amino acid sequence selected from an amino acid sequence of Necl-4 or fragments thereof.
- A second aspect of the invention relates to nucleic acid molecules encoding a polypeptide as defined above or fusion proteins as described below. Such nucleic acids also include vectors containing said molecules, in particular expression vectors.
- A third aspect of the invention resides in a fusion protein having inhibitory activity to a Necl protein such as Necl-1 or Necl-2 or Necl-3 or Necl-4 or a ligand thereof.
- A fourth aspect of the invention relates to antibodies that selectively bind the polypeptides according to the invention, as well as to antibodies that bind Necl-3 (Necl-4) and/or block the binding of Necl-3 (Necl-4) to other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4 and to a use of an antibody according to the invention in an assay.
- A fifth aspect of the invention relates to host cells expressing a polypeptide as defined above, as well as methods of producing such cells.
- A sixth aspect of the invention is a process for preparing a polypeptide or a fusion protein as defined above, typically using recombinant technologies.
- A seventh aspect of the invention relates to a pharmaceutical composition comprising a polypeptide or a nucleic acid as defined above and a pharmaceutically acceptable carrier or vehicle.
- An eighth aspect of the invention is a use of a Necl-3 antagonist and/or Necl-4 antagonist for the preparation of a medicament for the treatment of CNS disorders.
- A ninth aspect of the invention is a Necl-3 antagonist or a Necl-4 antagonist for use as a medicament.
- A tenth aspect of the invention provides a kit comprising at least one Necl-3 antagonist and/or Necl-4 antagonist according to the invention.
- An eleventh aspect of the invention relates to a method of treating of a disease comprising the administration of a therapeutically effective amount of a Necl-3 antagonist or a Necl-4 antagonist according to the invention in a mammal in need thereof and wherein the disease is a CNS disorders as well as methods of promoting remyelination of nerve cells in a mammal comprising administering to the mammal a Necl-3 and/or Necl-4 antagonist according to the invention.
- A twelfth aspect of the invention is a method of antagonizing the aggregation of Necl-3 and/or of Necl-4 with itself of with at least one protein of the Necl/SynCAM family.
- A thirteenth aspect is a method for in vitro detection of the level of a Necl-3 and of Necl-4 protein in a biological sample.
- A fourteenth aspect is a method for screening for a Necl-3 and/or Necl-4 antagonist.
- Other features and advantages of the invention will be apparent from the following detailed description.
-
- Figure 1 shows the nucleic acid sequence (SEQ ID NO: 1) and the amino acid sequence of human Necl-3 (SEQ ID NO: 2).
- Figure 2: Figure 2a shows the nucleic acid sequence (SEQ ID NO: 3) and Figure 2b shows the amino acid sequence of the extracellular fragment aa 1-365 of human Necl-3 (SEQ ID NO: 4).
- Figure 3 shows the nucleic acid sequence (SEQ ID NO: 5) and the amino acid sequence of human Necl-4 (SEQ ID NO: 6).
- Figure 4: Figure 4a shows the nucleic acid sequence (SEQ ID NO: 7) and Figure 4b shows the amino acid sequence of the extracellular fragment aa 1-323 of human Necl-4 (SEQ ID NO: 8).
- Figure 5: Figure 5a shows the nucleic acid sequence (SEQ ID NO: 14) and Figure 4b shows the amino acid sequence of the extracellular fragment aa 169-367 of human Necl-3 (SEQ ID NO: 15).
- Figure 6: Figure 6a shows the nucleic acid sequence (SEQ ID NO: 25) and Figure 6b shows the amino acid sequence of the extracellular fragment aa 126-322 of human Necl-4 (SEQ ID NO: 26).
- Figure 7: Figure 7a shows the amino acid sequence (SEQ ID NO: 35) of IgG1 and Figure 7b shows the amino acid sequence of IgG4 (SEQ ID NO: 36) of the fusion proteins according to the invention.
- The term "neurodegenerative" comprises a disease or a state characterized by a CNS degeneration or alteration, especially at the level of the neurons. It comprises neuro-inflammatory and demyelinating states or diseases such as leukoencephalopathies, and leukodystrophies.
- The term "demyelinating" is referring to a state or a disease of the CNS comprising the degradation of the myelin around the axons. In the context of the invention, the term demyelinating disease is intended to comprise conditions which comprise a process that demyelinate cells such as multiple sclerosis, progressive multifocal leukoencephalopathy (PML), myelopathies, any neuroinflammatory condition involving autoreactive leukocyte within the CNS, congenital metabolic disorder, a neuropathy with abnormal myelination, drug induced demyelination, radiation induced demyelination, a hereditary demyelinating condition, a prion induced demyelinating condition, encephalitis induced demyelination or a spinal cord injury. Preferably, the condition is multiple sclerosis.
- The term "multiple sclerosis" includes relapsing remitting MS (RRMS), Secondary Progressive MS (SPMS), primary progressive MS (PPMS). Patients suffering from MS can be defined for example as having clinically definite or laboratory-definite MS according to Schumacher or Poser criteria ( Schumacher et al., 1965, Ann. NYAcad. Sci. 1965; 122:552-568 ; Poser et al., 1983, Ann. Neurol. 13(3): 227-31 ).
- The term "Efficacy" of a treatment according to the invention can be measured based on changes in the course of disease in response to a use according to the invention. For example, the efficacy of a treatment of MS can be measured by the frequency of relapses in RRMS and the presence or absence of new lesions in the CNS as detected using methods such as MRI technique ( Miller et al., 1996, Neurology, 47(Suppl 4): S217 ; Evans et al., 1997, Ann. Neurology, 41:125-132 ). The observation of the reduction and/or suppression of MRI T1 gadolinium-enhanced lesions (thought to represent areas of active inflammation) gives a primary efficacy variable. Secondary efficacy variables include MRI T1 enhanced brain lesion volume, MRI T1 enhanced lesion number, MRI T2 lesion volume (thought to represent total disease burden, i.e. demyelination, gliosis, inflammation and axon loss), MRI T1 enhanced hypointense lesion volume (thought to represent primarily demyelination and axon loss), time-to-progression of MS, frequency and severity of exacerbations and time-to-exacerbation, Expanded Disability Status Scale score and Scripps Neurologic Rating Scale (SNRS) score ( Sipe et al., 1984, Neurology, 34, 1368-1372 ). Methods of early and accurate diagnosis of multiple sclerosis and of following the disease progression are described in Mattson, 2002, Expert Rev. Neurotherapeutics, 319-328 . Degree of disability of MS patients can be for example measured by Kurtzke Expanded Disability Status Scale (EDSS) score ( Kurtzke, 1983, Neurology, 33, 1444-1452 ). Typically a decrease in EDSS score corresponds to an improvement in the disease and conversely, an increase in EDSS score corresponds to a worsening of the disease. Any other clinical assessment of the disease known is available to the skilled person to measure the efficacy of the treatment according to the invention.
- "Relapses" involve neurological problems that occur over a short period, typically days but sometimes as short as hours or even minutes. These attacks most often involve motor, sensory, visual or coordination problems early in the disease. Later, bladder, bowel, sexual and cognitive problems may be shown. Sometimes the attack onset occurs over several weeks. Typical MS relapse involves a period of worsening, with development of neurological deficits, then a plateau, in which the patient is not getting any better but also not getting any worse followed by a recovery period. Recovery usually begins within a few weeks.
- As used herein, "treatment" and "treating" and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof and/or may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or relieving the disease, i.e., causing regression of the disease and/or its symptoms or conditions.
- The term "subject" as used herein refers to mammals. For examples, mammals contemplated by the present invention include human, primates, domesticated animals such as cattle, sheep, pigs, horses and the like.
- The term "Fc domain" encompasses native Fc, including the various subtypes, and Fc variant molecules and sequences as defined below. It includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
- The term "native Fc" refers to a molecule or sequence comprising the sequence of a non-antigen binding fragment resulting from the digestion of whole antibody, or by means of recombinant DNA techniques, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and may be any of the immunoglobulins. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e. disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e. g. IgG, IgA, IgE, IgM, IgD) or subclass (e.g. IgG1, IgG2, IgG3, IgG4, IgAl, IgA2). Examples of native Fc domains are human IgG1 and IgG4, having respectively the amino acid sequence of SEQ ID NO: 35 and of SEQ ID NO: 36. The term "native Fc" as used in the context of the invention is generic to the monomeric, dimeric, and multimeric forms.
- The term "Fc variant" refers to a molecule or sequence that is modified from a native Fc, for example a molecule or sequence that is humanized from a non-human native Fc. A native Fc comprises sites that may be removed or changed because they provide structural features or biological activities that are not required, or alternatively enhanced, for the fusion molecules of the present invention. Thus, the term "Fc variant" comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (i) disulfide bond formation, (ii) incompatibility with a selected host cell (iii) N-terminal heterogeneity upon expression in a selected host cell, (iv) glycosylation, (v) interaction with complement, (vi) binding to an Fc receptor other than a salvage receptor, or (vii) antibody-dependent cellular cytotoxicity (ADCC).
- The term "Fc fusion proteins" in general refers to molecules comprising at least part of an immunoglobulin Fc domain and at least one peptide. Such fusion proteins may be multimers or dimers or fragment thereof and may be derivatized. Suitable fusion proteins of the invention include a Necl-3 polypeptide e.g. an extracellular domain of Necl-3, a fragment thereof or a mutein thereof, linked to an immunoglobulin Fc region.
- Suitable fusion proteins of the invention include a Necl-4 polypeptide e.g. an extracellular domain of Necl-4, a fragment thereof or a mutein thereof, linked to an immunoglobulin Fc region. The extracellular domain, the fragment or the mutein thereof may be fused directly or through linker sequences to the Fc portion of an immunoglobulin. The preparation of fusion proteins comprising certain heterologous polypeptides fused to an Fc domain is known in the art
WO 91/08298 WO 96/08570 WO 93/22332 WO 04/085478 WO 01/03737 WO 02/66514 - The term "isolated" is used to indicate that the molecule is free of association with other proteins or polypeptides, for example as a purification product of recombinant host cell culture or as a purified extract.
- The term "antibody" comprises antibodies binding to Necl-3 or Necl-4 protein or fragment thereof, chimeric antibodies recognizing and/or binding selectively to Necl-3 or Necl-4 protein or fragment thereof, fully human, humanized, genetically engineered or bispecific or multispecific antibodies as well as fragments thereof such as single chain antibodies (scFv) or domain antibodies against Necl-3 and/or Necl-4 protein or fragment thereof and the like. Antibodies of this invention may be monoclonal or polyclonal antibodies, or fragments or derivative thereof having substantially the same antigen specificity. The term "selectively" indicates that the antibodies preferentially recognize and/or bind the target polypeptide or epitope, i.e., with a higher affinity than any binding to any other antigen or epitope, i.e. the binding to the target polypeptide can be discriminated from non-specific binding to other antigens. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis ( Scatchard et al., 1949, Ann NY Acad. Sci., 51, 660-672 ).
- The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
- The term "antagonists" is defined as a molecule that antagonizes completely or partially the activity of biological molecule.
- The term "peptidomimetic" is defined as a peptide analog containing non-peptidic structural elements, which peptide is capable of mimicking or antagonizing the biological action(s) of a natural parent peptide. A peptidomimetic does no longer have classical peptide characteristics such as enzymatically scissile peptide bonds.
- The nucleotide sequence encoding human Necl-3, isolated as described in Examples 1 and 2, is presented in SEQ ID NO: 1 and the amino acid sequence of human Necl-3 is described in SEQ ID NO: 2. The human Necl-3 amino acid sequence described in SEQ ID NO: 2 has a predicted extracellular domain spanning from
amino acids 1 to 367. - As used herein, the term Necl-3 encompasses polypeptides having an amino acid sequence of SEQ ID NO: 2 and fragments thereof such as the corresponding full length extracelullar domain (aa1-365) of SEQ ID NO: 4 or a fragment thereof (aa169-367) having an amino acid sequence of SEQ ID NO: 15. In addition, Necl-3 encompasses polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequence of SEQ ID NO: 2, or with the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence of SEQ ID NO: 15 and which polypeptides are biologically active. The term Necl-3 encompasses Necl-3 secreted peptide where the signal peptide is cleaved. Typically, the signal peptide is cleaved between
position 24 and 25 of the SEQ ID NO: 4 as determined by Signal P 3.0 software. - The nucleotide sequence encoding human Necl-4, isolated as described in Examples 6 and 7, is presented in SEQ ID NO: 5 and the amino acid sequence of human Necl-4 is described in SEQ ID NO: 6. The human Necl-4 amino acid sequence described in SEQ ID NO: 6 has a predicted extracellular domain of from
amino acids 1 to 323. - As used herein, the term Necl-4 encompasses polypeptides having an amino acid sequence of SEQ ID NO: 6 and fragments thereof such as the full length extracelullar domain (aal-323) of SEQ ID NO: 8 or a fragment thereof (aa126-322) having an amino acid sequence of SEQ ID NO: 26. In addition, Necl-4 encompasses polypeptides that have a high degree of similarity or a high degree of identity with the amino acid sequence of SEQ ID NO: 6 or with the amino acid sequence of SEQ ID NO: 8 or with the amino acid sequence of SEQ ID NO: 26 and which polypeptides are biologically active.
- The term Necl-4 encompasses Necl-4 secreted peptide where the signal peptide is cleaved. Typically, the signal peptide is cleaved between
position 24 and 25 of the SEQ ID NO: 8 as determined by Signal P 3.0 software. - A "Necl-3 variant" or a "Necl-4 variant" as referred to herein, means a polypeptide substantially homologous to native Necl-3 or native Nelc-4, respectively, but which has an amino acid sequence different from that of native Necl-3 or Necl-4 respectively (human, murine or other mammalian species) because of one or more deletions, insertions or substitutions. Substantially homologous means a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the native amino acid sequences, as disclosed above. The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and/or mathematical calculation, or more easily by comparing sequence information using a computer program such as Clustal package version 1.83. Necl-3 variants or Necl-4 variants may comprise a sequence having at least one conservatively substituted amino acid, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics. Generally, substitutions for one or more amino acids present in the native polypeptide should be made conservatively. Examples of conservative substitutions include substitution of amino acids outside of the active domain(s), and substitution of amino acids that do not alter the secondary and/or tertiary structure of Necl-3 or Necl-4 respectively. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known ( Kyte, et al., 1982, J. Mol. Biol., 157: 105- 131 ). Naturally occurring variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the native protein, wherein the native biological property is retained. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non native residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. Exemplary amino acid substitutions are presented in Table 1 below:
Table 1 Original residues Examples of substitutions Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln Asp (D) Glu Cys (C) Ser, Ala Gln (Q) Asn Glu (E) Asp Gly (G) Pro, Ala His (H) Asn, Gln, Lys, Arg Ile (I) Leu, val, Met, Ala, Phe, Norleucine Leu (L) Ile, Val, Met, Ala, Phe, Norleucine Lys (K) Arg, Gln, Asn Met (M) Leu, Ile, Phe Phe (F) Leu, Val, Ile, Ala, Tyr Pro (P) Ala, Gly Ser (S) Thr, Ala, Cys Thr (T) Ser Trp (W) Tyr, Phe Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Met, Leu, Phe, Ala, Norleucine - Further examples ofNecl-3 or Necl-4 are provided herein and include but is not limited to variants, biologically active fragments, fragments and fragments of variants. Necl-3 variants or Necl-4 variants may be used as antagonists of Necl-3 activity or Necl-4 activity or be used to develop Necl-3 antagonists or Necl-4 antagonists, such as antibodies, chimaeric proteins or fusion proteins according to the invention.
- The term "Necl-3 antagonist" comprises all antagonists of all suitable forms of Necl-3 described herein that antagonize one or more biological activity of Necl-3 and of Necl-3 variants or fragment thereof. For example, the Necl-3 antagonists of the invention are able to antagonize the ability of Necl-3 to participate to homophilic interactions (interactions between Necl-3 proteins) and/or heterophilic interactions (interactions with other protein(s) from the Necl or synCAM family such as Necl-1, Necl-2 and Necl-4, or with other membrane protein ligands). The term "Necl-3 antagonist" includes but is not limited to: Necl-3 specific antibodies of any sort (polyclonal, monoclonal, antibody fragments, antibody variants), chimaeric proteins, natural or unnatural proteins with Necl-3 antagonizing activities, small molecules, nucleic acid derived polymers (such as DNA and RNA aptamers, PNAs, or LNAs), peptidomimetics, fusion proteins, or gene therapy vectors driving the expression of such Necl-3 antagonists. Further embodiments include as Necl-3 antagonists, soluble Necl-3 fusion proteins such as but not limited to an extracellular domain of Necl-3 or a fragment thereof linked to an Fc domain. Typically, Necl-3 antagonists are able to bind Necl-3 and/or to block the binding of Necl-3 and other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4.
- The term "Necl-4 antagonist" comprises all antagonists of all suitable forms of Necl-4 described herein that antagonize one or more biological activity of Necl-4 and of Necl-4 variants. For example, the Necl-4 antagonists of the invention are able to antagonize the ability of Necl-4 to participate to homophilic interactions (interactions between Necl-4 proteins) and/or heterophilic interactions (interactions with other protein(s) from the SynCAM family such as Necl-1, Necl-2 and Necl-3, or with other membrane protein ligands). The term Necl-4 antagonist includes but is not limited to: Necl-4 specific antibodies, chimeric proteins, small molecules, peptidomimetics and fusion proteins. Further embodiments include as Necl-4 antagonists, soluble Necl-4 fusion proteins such as but not limited to an extracellular domain of Necl-4 or a fragment thereof linked to an Fc domain. Typically, Necl-4 antagonists are able to bind Necl-3 and/or to block the binding of Necl-4 and other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4.
- The biological consequences of the homophilic and heterophilic interactions referred in the context of the invention for instance lead to cell-cell contact, to cell movement, to cell aggregation or to cellular activation.
- An antibody that is immunoreactive with Necl-3 and/or especially to an extracellular fragment of Necl-3 or a fragment thereof e.g. to an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 15 may be used as a Necl-3 antagonist.
- An antibody that is immunoreactive with Necl-4 and/or especially to an extracellular fragment of Necl-4 or a fragment thereof e.g. to an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 26 may be used as a Necl-4 antagonist.
- A Necl-3 or a Necl-4 protein, a Necl-3 antagonist or a Necl-4 antagonist, as an isolated, purified or homogeneous protein according to the invention, may be produced by recombinant expression systems as described herein or purified from naturally occurring cells.
- Suitable expression of Necl-3, Necl-3 variants or fragments, Necl-3 antagonists, Necl-4, Necl-4 variants or fragments and Necl-4 include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cellular hosts are described for example in Pouwels et al., 1985, Cloning Vectors: A laboratory manual, Elsevier New York .
- Prokaryotes include gram negative and gram positive organism such as E. Coli or Bacilli. Suitable prokaryotic host cells include for example E. Coli BL21 strain. In prokaryotic host cells, such as E. coli, a Necl-3, Necl-3 fragment or variant, Necl-3 antagonist, Necl-4, Necl-4 fragment or variant or Necl-4 antagonist may include a N-terminal methionine residue to facilitate the expression of recombinant polypeptide in the prokaryotic host cell. The N-terminal Met may be cleaved from the expressed peptide.
- The invention provides pharmaceutical or therapeutic agents as compositions and methods for treating a patient, preferably a mammalian patient, and most preferably a human patient who is suffering from a medical disorder, and in particular a disorder mediated by Necl-3 and/or Necl-4, such as neuroinflammatory disorders and/or neurodegenerative disorders.
- Pharmaceutical compositions of the invention can contain one or more Necl-3 antagonist and/or Necl-4 antagonist (including from recombinant and non-recombinant sources) in any form described herein. Compositions of this invention may further comprise one or more pharmaceutically acceptable additional ingredient(s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.
- The compounds of the invention, together with a conventionally employed adjuvant, carrier, diluent or excipient may be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed. Compositions according to the invention are preferably injectable.
- Compositions of this invention may also be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. The compositions may also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Further materials as well as processing techniques and the like are set out in
Part 5 of Remihgtoh's Pharmaceutical Sciences, 20th Edition, 2000, Marck Publishing Company, Easton, Pennsylvania - Solid compositions of this invention may be in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to, syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, maizestarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycollate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets may be coated according to methods well known in the art.
- Injectable compositions are typically based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art.
- Compositions of this invention may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides. Compositions of this invention may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane. Compositions of this invention may also be formulated transdermal formulations comprising aqueous or non-aqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
- Compositions of this invention may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents. The composition may also be provided in a powder form for reconstitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.
- Compositions of this invention may also be formulated as a depot preparation, which may be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).
- Compositions of this invention may also be formulated as a liposome preparation. The liposome preparation can comprise liposomes which penetrate the cells of interest or the stratum corneum, and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell. Other suitable formulations can employ niosomes. Niosomes are lipid vesicles similar to liposomes, with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
- The compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can also be found in the incorporated materials in Remington's Pharmaceutical Sciences.
- Compositions of this invention may be administered in any manner including, but not limited to, orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal or intranasal administration, or combinations thereof Parenteral administration includes, but is not limited to, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intra-thecal, and intra-articular. The compositions of this invention may also be administered in the form of an implant, which allows slow release of the compositions as well as a slow controlled i.v. infusion. In a preferred embodiment, a Necl-3, or a Necl-3 antagonist, or a necl-4 or a Necl-4 antagonist according to the invention are administered intravenously or subcutaneously.
- This invention is further illustrated by the following examples that are not intended to limit the scope of the invention in any way.
- The dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
- According to the invention, a Necl-3 antagonist and/or a Necl-4 antagonist according to the invention can be administered alone or in combination with a co-agent useful in the treatment of CNS disorders including neuroinflammation and/or neurodegeneration, such as substances useful in the treatment of demyelinating diseases e.g. for example a co-agent selected from IFN-beta, Glatiramer acetate, Corticosteroids, immunusuppressants such as Mitoxantrone, Azathioprine, Cyclophosphamide, Methotrexate, Natalizumab (Tysabri®), metalloproteinases inhibitors, especially MMP-12 and/or MMP-9 inhibitors such as Minocycline.
- The invention encompasses the administration of a Necl-3 antagonist or Necl-4 antagonist of the invention wherein the Necl-3 antagonist or the Necl-4 antagonist is administered to an individual prior to, simultaneously or sequentially with other therapeutic regimens or co-agents useful in the treatment of neuroinflammation and/or neurodegeneration (e.g. multiple drug regimens), in a therapeutically effective amount. The Necl-3 antagonist and/or a Necl-4 antagonist according to the invention that are administered simultaneously with said co-agents can be administered in the same or different compositions and in the same or different routes of administration.
- The term "interferon-beta (IFN-β)", as used herein, is intended to include fibroblast interferon in particular of human origin, as obtained by isolation from biological fluids or as obtained by DNA recombinant techniques from prokaryotic or eukaryotic host cells, as well as its salts, functional derivatives, variants, analogs and active fragments.
- IFN-β suitable in accordance with the present invention is commercially available e.g. as Rebif® (Serono), Avonex® (Biogen) or Betaferon® (Schering). The use of interferons of human origin is also preferred in accordance with the present invention. The term interferon, as used herein, is intended to encompass salts, functional derivatives, variants, analogs and active fragments thereof
- In an embodiment, patients according to the invention are patients suffering from CNS disorders such as neuroinflammatory and neurodegenerative disorders or states.
- In another embodiment, patients according to the invention are patients suffering from demyelinating states or disorders.
- In a further embodiment, patients according to the invention are patients suffering from multiple sclerosis.
- The nucleic acids encoding Necl-3, Necl-3 antagonists, Necl-4, Necl-4 antagonists and fragments thereof may be used to express recombinant polypeptides for analysis, characterization and therapeutic use.
- Necl-3 antagonists and Necl-4 antagonists according to the invention are useful in the treatment of CNS disorders, including neuroinflammatory and/or neurodegenerative disorders or conditions such as demyelinating diseases or conditions.
- Necl-3 antagonists according to the invention are useful to antagonize one or more biological activity of Necl-3 such as adhesion properties, including Necl-3 induced cell adhesion processes, for example adhesion process between Necl-3 on myelinated axons and cells of the immune system expressing Necl-3 or Necl-3 heterophilic ligands.
- Necl-4 antagonists according to the invention are useful to antagonize one or more biological activity of Necl-4 such as adhesion properties, including Necl-4 induced cell adhesion processes, for example adhesion process between Necl-4 on astrocytes and cells of the immune system expressing Necl-4 or Necl-4 heterophilic ligands
- The disclosed Necl-3 nucleic acid sequences or Necl-4 nucleic acid sequences, or fragments thereof and combinations of fragment thereof may be used as probes or primers.
- The disclosed Necl-3 or Necl-4 amino acid sequences, variants thereof, fragments thereof and combinations thereof may be used in a process for the preparation of Necl-3 antagonists and/or Necl-4 antagonists according to the invention.
- The disclosed Necl-3 antibodies and/or Necl-4 antibodies may be used in assays relating for example to the analysis of Necl-3 and/or Necl-4 expression such as western blots, immunohistochemistry, ELISA or FACS assays.
- Within the context of this invention, the beneficial effect includes but is not limited to an attenuation, reduction, decrease or diminishing of the pathological development after onset of the disease.
- Table 2 below presents the Sequence identity numbers and associated molecules:
Table 2 SEQ ID NO. Molecule 1 DNA sequence for human Necl-3 2 Amino acid sequence for human Necl-3 3 DNA sequence for full extracellular domain of human Necl-3 (1-365) 4 Amino acid sequence for full extracellular domain of human Necl-3 (1-365) 5 DNA sequence for human Necl-4 6 Amino acid sequence for human Necl-4 7 DNA sequence for full extracellular domain of human Necl-4 (1-323) 8 Amino acid sequence for full extracellular domain of human Necl-4 (1-323) 9 cDNA of clone DKFZp761G128 10 PCR primer for Necl-3 construct 11 PCR primer for Necl-3 construct 12 PCR primer for Necl-3 fragment (169-367) and (1-365) constructs 13 PCR primer for Necl-3 fragment (169-367) and (1-365)constructs 14 Nucleic acid sequence for a Necl-3 fragment (169-367) 15 Amino acid sequence for a Necl-3 fragment (169-367) 16 RT PCR primer for Necl-3 17 RT PCR primer for Necl-3 18 RT PCR primer for GAPDH 19 RT PCR primer for GAPDH 20 Modified cDNA of clone IMAGE 4375136 21 PCR primer for Necl-4 construct 22 PCR primer for Necl-4 construct 23 PCR primer for Necl-4 fragment (126-322) construct 24 PCR primer for Necl-4 fragment (126-322) construct 25 Nucleic acid sequence for a Necl-4 fragment (126-322) 26 Amino acid sequence for a Necl-4 fragment (126-322) 27 RT PCR primer for Necl-4 28 RT PCR primer for Necl-4 29 cDNA of clone IMAGE:5199627 30 PCR primer for Necl-1 construct 31 PCR primer for Necl-1 construct 32 cDNA of clone IMAGE:4701395 33 PCR primer for Necl-2 construct 34 PCR primer for Necl-2 construct 35 Amino acid sequence for Human IgG1 36 Amino acid sequence for Human IgG4 37 Amino acid sequence for Mouse IgG2a 38 Amino acid sequence for Mouse IgG3 39 Amino acid sequence for Necl-1 40 Amino acid sequence for Necl-2 41 Amino acid sequence for MOG peptide - One process for producing Necl-3 antagonists or Necl-4 antagonists according to the invention comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes a Necl-3 antagonist or a Necl-4 antagonist under conditions sufficient to promote expression of Necl-3 antagonists or Necl-4 antagonists, respectively.
- A Necl-3 antagonist or a Necl-4 antagonist according to the invention is then recovered from culture medium or cell extracts, depending upon the expression system employed. As known to the skilled artisan, procedures for purifying a recombinant protein will vary according to such factors as the type of host cells employed and whether or not the recombinant protein is secreted into the culture medium.
- For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
- Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange and/or an affinity resin can be employed. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media can be employed to further purify Necl-3, Necl-3 variants and Necl-3 antagonists and/or Necl-4, Necl-4 variants and Necl-4 antagonists. Some or all of the foregoing purification steps, in various combinations, are well known and can be employed to provide a substantially homogeneous recombinant protein.
- Recombinant protein produced in bacterial culture can be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps.
- Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
- A desired DNA sequence may be chemically synthesized using techniques known per se. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. Linkers containing restriction endonuclease cleavage site(s) may be employed to insert the desired DNA fragment into an expression vector, or the fragment may be digested at cleavage sites naturally present therein. The well known polymerase chain reaction procedure also may be employed to amplify a DNA sequence encoding a desired protein fragment. As a further alternative, known mutagenesis techniques may be employed to insert a stop codon at a desired point, e. g. immediately downstream of the codon for the last amino acid of the receptor-binding domain.
- Typically, when the Necl-3 and/or Necl-4 antagonists are fusion proteins (Fc-Necl-3 and Fc-Necl-4 fusion protein) according to the invention, they may be prepared according to known processes: for example, the nucleic acid sequence encoding the extracellular domain of Necl-3 or Necl-4 or a fragment thereof can be cloned in an expression vector fused to a nucleic acid sequence encoding the original extracellular domain of Necl-3 or Necl-4 or a fragment thereof signal sequence (or any other appropriate signal/export sequence) at its 5' end, and the nucleic acid sequence encoding the constant region of human immunoglobulin lambda heavy chain IgG (e.g. IgG1 or IgG4) at its 3'end. The resulting vector can be used to transform a mammalian (e.g. CHO or HEK293) or an insect (e.g. Schneider S2 cells) host cell line and the clones stably expressing and secreting the recombinant fusion protein having extracellular domain of Necl-3 or Necl-4 or a fragment thereof at the N-terminus and the IgG sequence at the C-terminus can be selected. This clone then can be used for scaling up the production and for purifying the recombinant fusion protein from the culture medium. Alternatively, the position of the nucleic acid encoding the constant region of human immunoglobulin lambda heavy chain IgG and extracellular domain of Necl-3 or Necl-4 or a fragment thereof can be inversed, and the resulting protein can be expressed and secreted using still the original signal sequence of extracellular domain of Necl-3 or Necl-4 or a fragment thereof, or any other appropriate signal/export sequence. Using these technologies it can be also possible to generate heterodimers if two different constructs expressing one Necl-3-Fc or Necl-4-Fc fusion protein and the other a different Fc-based fusion protein (for example another antagonist) are coexpressed in the same host cell (
WO 00/18932 - Typically, preparation of the fusion protein according to the invention in insect cells is carried out by the co-transfection of the corresponding insect cell expression vector expressing Fc-Necl-3 extra-cellular domain or Fc-Necl-4 extra-cellular domain (such as illustrated in Examples 6 and 12) along with the plasmid pCoBlast that carries a blasticidin S resistance gene and stable clones are selected with blasticidin S according to methods commonly known in the art. Of these, high expressers of the fusion proteins are selected.
- Alternatively, the preparation in mammalian cells (CHO, BHK, COS, HEK-293 or other cell line) is carried out by the transfected of the corresponding mammalian cell expression vector expressing Fc-Necl-3 extra-cellular domain or Fc-Necl-4 (such as illustrated in Examples 6 and 12) and stable clones are selected with Zeocin according to methods commonly known in the art. Of these, high expressers of the fusion proteins are selected.
- Once selected, high expresser clones obtained from one of these methods are expanded and grown (e.g. in a 150 mL flat bottom flask). For instance, S2 insect cells are induced with CuSO4 (e.g. 0.5 mM). After several days (e.g. 4 days), the conditioned culture medium is loaded onto a Protein A Sepharose column and the Fc-Necl-3 or Fc-Necl-4 fusion protein is recovered according to general methods.
- Typically, when the Necl-3 and/or Necl-4 antagonists are antibodies to Necl-3/Necl-4 according to the invention, they may be prepared according to known processes.
- Methods of preparing polyclonal antibodies from various species have been described for instance in Vaitukaitis et al., 1971, J Clin Endocrinol Metab., 33, 988 . Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include Necl-3 or Necl-4 or the extracellular fragment or a fragment thereof or a variant as described here above or a fusion protein thereof, or a synthetic peptide encoding a region of Necl-3 or Necl-4. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separated.
- Methods of producing monoclonal antibodies may be found, for instance, in Kohler et al., 1975, Nature, 256, 495 incorporated therein by reference in its entirety.
- The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in
US 4,816,567 . - Typically, the preparation of the polyclonal antibodies according to the invention is performed by using a fragment of the extracellular domain of Necl-3 or Necl-4 (e.g. the Necl-3(aa169-367) fragment of SEQ ID NO: 15 or Necl-4(aa126-322) of SEQ ID NO: 26) produced in E. coli to immunize rabbits. The same fragment is coupled to NHS activated Sepharose 4 Fast Flow and used for affinity purification of the antibodies from the rabbit sera. The antibody-containing fractions are then loaded onto a Protein A Sepharose CL-4B column and the IgGs are eluted.
- Typically, the efficacy of the Necl-3/Necl-4 antagonists according to the invention may be assayed in various cell adhesion assays such as described in Biederer et al., 2002, Science. 297:1525-31 or according to the following general process: S2 Drosophila cells are maintained in appropriate medium such as Schneider's Drosophila Medium (Gibco) supplemented with 10% FCS and transfected with an appropriate transfection agent (e.g. Lipofectamine 2000 (Invitrogen), Fugene HD (Roche), or other). After transfection (e.g. about 24 hours) the medium is changed and CuSO4 is added (e.g. to 0.5 mM) for another period of time (e.g. 24 hours). For aggregation assays, the cells to be tested are mixed after the medium change, induced with CuSO4 and gently shaken for several hours to allow for plasmid transcription, protein accumulation and aggregate formation. The Necl-3/Necl-4 antagonists and appropriate controls such as unrelated Fc fusion proteins or immunoglobulins are added prior to induction with CuSO4 and their inhibitory effect on aggregate formation is assessed by microscopy.
- In one embodiment, the invention provides an isolated polypeptide having at least 80% (such as at least 85%, at least 90%, at least 95%, at least 98%) identity or homology with a sequence of amino acids selected from the group consisting of:
- (a)
amino acids 1 to 435 of SEQ ID NO: 2; - (b)
amino acids 1 to 365 SEQ ID NO: 4; - (c)
amino acids 1 to 388 of SEQ ID NO: 6; - (d)
amino acids 1 to 323 of SEQ ID NO: 8; - (e)
amino acids 1 to 198 of SEQ ID NO: 15; and - (f)
amino acids 1 to 197 of SEQ ID NO: 26. - In a further embodiment, the invention provides an isolated polypeptide according to the invention wherein the isolated polypeptide binds to at least one Necl protein or one of its ligand such as Necl-1, Necl-2, Necl-3 or Nec-4 or fragment thereof In a particular embodiment, the isolated polypeptide according to the invention binds to the extracellular fragment or a fragment thereof of at least one Necl protein, such as Necl-1, Necl-2, Necl-3 or Nec-4.
- In a further embodiment, the invention provides an isolated polypeptide according to the invention comprising an amino acid sequence selected from the group consisting of:
- SEQ ID NO: 2; SEQ ID NO: 4 and SEQ ID NO: 15. In a further embodiment, the isolated peptide comprises an amino acid sequence of SEQ ID NO: 4.
- In another further embodiment, the invention provides an isolated polypeptide according to the invention comprising an amino acid sequence selected from the group consisting of:
- SEQ ID NO: 6; SEQ ID NO: 8 and SEQ ID NO: 26. In a further embodiment, the isolated peptide comprises an amino acid sequence of SEQ ID NO: 8.
- In another embodiment, the invention provides an isolated nucleic acid consisting of a nucleotide sequence encoding a polypeptide having at least 80% (such as at least 85%, at least 90%, at least 95%, at least 98%) identity or homology with a sequence of amino acids selected from the group consisting of:
- (a)
amino acids 1 to 435 of SEQ ID NO: 2; - (b)
amino acids 1 to 365 SEQ ID NO: 4; - (c)
amino acids 1 to 388 of SEQ ID NO: 6; - (d)
amino acids 1 to 323 of SEQ ID NO: 8; - (e)
amino acids 1 to 198 of SEQ ID NO: 15; and - (f)
amino acids 1 to 197 of SEQ ID NO: 26. - In a further embodiment, the invention provides an isolated nucleic acid consisting of a nucleotide sequence encoding a polypeptide having a sequence of amino acids selected from the group consisting of:
- (a)
amino acids 1 to 435 of SEQ ID NO: 2; - (b)
amino acids 1 to 365 of SEQ ID NO: 4; - (c)
amino acids 1 to 388 of SEQ ID NO: 6; - (d)
amino acids 1 to 323 of SEQ ID NO: 8; - (e)
amino acids 1 to 198 of SEQ ID NO: 15; and - (f)
amino acids 1 to 197 of SEQ ID NO: 26. - In another embodiment, the invention provides a fusion protein comprising the following sequences:
- an effector region having at least 80% identity or homology (such as at least 85%, at least 90%, at least 95%, at least 98%) with a sequence of amino acids selected from the group consisting of an extracellular domain of said Necl-3 or Necl-4 or a fragment thereof and
- an immunoglobulin constant region (Fc domain or an Fc variants thereof).
- In another embodiment, the invention provides a fusion protein comprising the following sequences:
- an effector region having at least 80% identity or homology (such as at least 85%, at least 90%, at least 95%, at least 98%) with a sequence of amino acids selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 15; and
- an immunoglobulin constant region (Fc domain or an Fc variants thereof).
- In another embodiment, the invention provides a fusion comprising the following sequences:
- an effector region having at least 80% identity or homology (such as at least 85%, at least 90%, at least 95%, at least 98%) with a sequence of amino acids of SEQ ID NO: 8 and SEQ ID NO: 26; and
- an immunoglobulin constant region (Fc domain or an Fc variants thereof).
- In another further embodiment, the invention provides a fusion protein according to the invention wherein the effector region is a protein with a sequence of amino acid of SEQ ID NO:4.
- In another further embodiment, the invention provides a fusion protein according to the invention wherein the effector region is a protein with a sequence of amino acid of SEQ ID NO:8.
- In a further embodiment, the invention provides a fusion protein according to the invention wherein the immunoglobulin constant region is the Fc domain of an IgG.
- In another further embodiment, the invention provides a fusion protein according to the invention wherein the Fc domain is the Fc domain of an IgG1 or an IgG4.
- In another further embodiment, the invention provides a fusion protein according to the invention wherein the fusion protein is selected from the following group:
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 169-367) of SEQ ID NO: 15 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the mouse Fc domain of an IgG2a of SEQ ID NO: 37;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the mouse Fc domain of an IgG3 of SEQ ID NO: 38;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the mouse Fc domain of an IgG2a of SEQ ID NO: 37; and
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the mouse Fc domain of an IgG3 of SEQ ID NO: 38.
- In another further embodiment, the invention provides a fusion protein according to the invention wherein the fusion protein is selected from the following group:
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35; and
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36.
- In a particular embodiment, the fusion protein according to the invention of the invention has an inhibitory activity to a Necl protein such as Necl-1 or Necl-2 or Necl-3 or Necl-4 or a ligand thereof.
- In another embodiment, the invention provides an isolated DNA sequence that encodes a fusion protein according to the invention.
- In a further embodiment, the invention provides an isolated antibody or an immunologically active fragment of a monoclonal antibody that binds to at least one Necl protein selected from Necl-3 and Necl-4.
- In another further embodiment, the invention provides an isolated antibody specifically directed against a polypeptide having a sequence of amino acids selected from the group consisting of:
- (a)
amino acids 1 to 435 of SEQ ID NO: 2; - (b)
amino acids 1 to 365 of SEQ ID NO: 4; - (c)
amino acids 1 to 388 of SEQ ID NO: 6; - (d)
amino acids 1 to 323 of SEQ ID NO: 8; - (e)
amino acids 1 to 198 of SEQ ID NO: 15; and - (f)
amino acids 1 to 197 of SEQ ID NO: 26. - In another embodiment, the invention provides a use of an antibody according to the invention for the analysis of Necl-3 or Necl-4 expression such as in a western blot, an immunohistochemistry, an ELISA or a FACS assay.
- In another embodiment, the invention provides a kit comprising at least one Necl-3 antagonist and/or Necl-4 antagonist according to the invention. More especially, the invention provides a kit comprising at least one Necl-3 antagonist and/or Necl-4 antagonist according to the invention wherein the Necl-3 antagonist and/or Necl-4 antagonist is a fusion protein according to the invention. The kit according to the invention may be used for measuring the activity/or the presence of at least one Necl protein such as Necl-1, Necl-2, Necl-3 or Necl-4 or its ligand. In a particular embodiment, the kit is used for measuring the activity/or the presence of Necl-3 and/or Necl-4.
- In another embodiment, the invention provides a recombinant expression vector comprising a nucleic acid molecule according to the invention, wherein the vector optionally comprises an expression control sequence, allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide, operably linked to the nucleic acid molecule.
- In another embodiment, the invention provides a host cell (e.g. prokaryotic or eukaryotic) transfected or transformed with a recombinant expression vector or a nucleic acid according to the invention.
- In another embodiment, the invention provides a process for producing cells capable of expressing a polypeptide according to the invention, comprising genetically engineering cells with a vector or a nucleic acid according to the invention
- In another embodiment, the invention provides a process for producing a polypeptide according to the invention, comprising transfecting an expression vector according to the invention into a host cell according to the invention under conditions allowing the expression of said polypeptide according to the invention and recovering the said polypeptide.
- In another embodiment, the invention provides a process for producing a fusion protein according to the invention, comprising culturing a host cell transformed with an expression vector according to the invention under conditions that promotes expression of said fusion protein and recovering said fusion protein.
- In another embodiment, the invention provides a pharmaceutical composition comprising a Necl-3 antagonist or a Necl-4 antagonist according to the invention, and a physiologically acceptable carrier, diluent or excipient.
- In particular, the invention provides a pharmaceutical composition wherein the Necl-3 antagonist or the Necl-4 antagonist is selected from a fusion protein according to the invention and a Necl-3 or a Necl-4 specific binding agent of the invention and a physiologically acceptable carrier, diluent or excipient. More especially, the invention provides a pharmaceutical composition comprising a Necl-3 antagonist or a Necl-4 antagonist according to the invention, wherein the Necl-3 antagonist or Necl-4 antagonist is a fusion protein according to the invention, and a physiologically acceptable carrier, diluent or excipient.
- In another embodiment, the invention provides a Necl-3 antagonist or a Necl-4 antagonist for use as a medicament. In particular, the Necl-3 antagonist or the Necl-4 antagonist according to the invention for use as a medicament is selected from a fusion protein according to the invention, an isolated Necl-3 specific binding agent or a Necl-4 specific binding agent according to the invention, and an antibody according to the invention.
- In another embodiment, the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases. In particular, the Necl-3 antagonist or the Necl-4 antagonist according to the invention is selected from a fusion protein according to the invention, an isolated Necl-3 specific binding agent or a Necl-4 specific binding agent according to the invention, and an antibody according to the invention.
- In a further embodiment, the Necl-3 antagonist or the Necl-4 antagonist according to the invention is a fusion protein according to the invention.
- In a further embodiment, the Necl-3 antagonist or the Necl-4 antagonist according to the invention is an antibody or an immunologically active fragment of a monoclonal antibody that binds Necl-3 (Necl-4) and/or blocks the binding of Necl-3 (Necl-4) to other binding partners such as Necl-1, Necl-2, Necl-3 and/or Necl-4
- In another embodiment, the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases wherein the Necl-3 antagonist or the Necl-4 antagonist is to be administered in combination with a co-agent that useful in the treatment of CNS disorders, including neuroinflammation and/or neurodegeneration such as a co-agent useful in the treatment of demyelination. In a particular embodiment, the Necl-3 antagonist or a Necl-4 antagonist according to the invention and the co-agent are used simultaneously or sequentially.
- In a further embodiment, the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of multiple sclerosis.
- In a further embodiment, the invention provides a use of a Necl-3 antagonist or a Necl-4 antagonist according to the invention for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases, wherein the Necl-3 antagonist or Necl-4 antagonist is a fusion protein according to the invention.
- In another embodiment, the invention provides a method of treating of a disease comprising the administration of a therapeutically effective amount of a Necl-3 antagonist or a Necl-4 antagonist according to the invention in a mammal in need thereof and wherein the disease is a CNS disorders comprising neuroinflammatory and neurodegenerative conditions or diseases such as demyelinating disorders or diseases. In a further embodiment, the mammal is human. In another further embodiment, the human suffers from multiple sclerosis.
- The invention relates to methods of promoting remyelination of nerve cells in a mammal comprising administering to the mammal a Necl-3 and/or Necl-4 antagonist according to the invention in a remyelination effective amount. In a particular embodiment, the mammal in the methods of the present invention is a human and the human suffers from a condition that demyelinates cells.
- In another embodiment, the invention provides a method of antagonizing the aggregation of Necl-3 and/or of Necl-4 with itself of with at least one protein of the Necl/SynCAM family, comprising exposing cells that express Necl-3 or Necl-4, respectively to a Necl-3 antagonist and/or Necl-4 antagonist according to the invention, such that the Necl-3 antagonist or the Necl-4 antagonist blocks the aggregation of Necl-3 or of Necl-4 with itself or with at least one protein of the Necl/SynCAM family. More particularly, the Necl-3 antagonist or Necl-4 antagonist used in this method is a fusion protein according to the invention.
- In another embodiment, the invention provides a method for in vitro detection of the level of a Necl-3 and of Necl-4 protein in a biological sample comprising contacting said biological sample with an antibody according to the invention.
- In another embodiment, the invention provides a method for screening for a Necl-3 and/or a Necl-4 antagonist, comprising the following steps:
- (i) Combining cells that do not aggregate spontaneously but in presence of a Necl-3 and/or a Necl-4 protein with at least one peptide according to the invention, in presence/in absence of a compound to be screened;
- (ii) Determining the ability of the compound to inhibit the Necl-3 and/or Necl-4 induced cell aggregation.
- References cited herein are hereby incorporated by reference in their entirety. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
- The invention having been described, the following examples are presented by way of illustration, and not limitation.
- The following abbreviations refer respectively to the definitions below:
- aa (amino acid); Da (Dalton), Kb (Kilobase), µl (microliter), mL (milliliter), mM (millimolar), min (minute), mW (MilliWatt), nm (nanometer), APC (Antigen Presenting Cell), BSA (Bovine Serum Albumin), CAM (Cell Adhesion Molecule), CHO (Chinese Hamster Ovary), CNS (Central nervous system), CSF (Cerebrospinal fluid), DMEM (Dulbecco's Modified Eagle Medium), DsRed2 (Discosoma Red), EDSS (Expanded Disability Status Scale), FACS (Fluorescence Activated Cell Sorte); FCS (Fetal calf serum), GADPH (Glyseraldehyde-3-phosphate dehydrogenase), GFP (Green Fluorescent Protein), IFN (interferon), Ig (Immunoglobulin), IPTG (isopropyl-beta-D-thiogalactopyranoside), i.v. (intra-venous), MBP (myelin basic protein), MHC (Major Histocompatibility Complex), MRI (Magnetic resonance imaging), MS (multiple sclerosis), NHS (N-hydroxysuccinimide), PCR (Polymerase Chain Reaction), PML (progressive multifocal leukoencephalopathy), PPMS (Primary progressive multiple sclerosis), PRMS (Progressive relapsing multiple sclerosis), RIPA (Radioimmunoprecipitation assay), RRMS (Relapsing-remitting multiple sclerosis), SDS (Sodium Dodecyl Sulfate), SNRS (Scripps Neurologic Rating Scale), SPMS (Secondary progressive multiple sclerosis), SynCAM (Synaptic CAM), s.c. (subcutaneous).
- Amplifications were performed by PCR reaction with Pfu Turbo polymerase (Stratagene) according to the supplier's instructions (1X cloned Pfu buffer, 200 µM each dNTP, 0.1-0.5 µM each primer, 2.5 units cloned enzyme, 1-100 ng plasmid template DNA, or 100-250 ng genomic template DNA).
- Mammalian expression vectors pNecl-3-GFP and pNecl3-DsRed2 were constructed as follows:
- the cDNA DKFZp761G128 (accession AL834270) of SEQ ID NO: 9 was used as a template for PCR amplification with the primers 5'-GGAATTCATGATTTGGAAACGC-3' of SEQ ID NO: 10 and 5'-CCCCAATTGCAATGAAATACTCTTT-3 of SEQ ID NO: 11, respectively.
- the resulting amplicon was digested with EcoR1 and Mfe1 and ligated into the EcoR1 site of pEGFP-N1 and pDsRed2-N1 (BD Biosciences Clonetech).
- Another mammalian expression vector pNecl-3-myc was constructed as follows:
- the cDNA DKFZp761G128 of SEQ ID NO: 9 was used as a template for PCR amplification with the primers 5'-GGAATTCATGATTTGGAAACGC-3' of SEQ ID NO: 10 and 5'-CCCCAATTGCAATGAAATACTCTTT-3 of SEQ ID NO: 11.
- the resulting amplicon was digested with EcoR1 and Mfe1 and ligated into the EcoR1 site of pcDNA3.1/myc-His (Invitrogen).
- The insect cell expression vector pMT-Necl-3-GFP and pMT-Necl-3-DsRed2 were constructed as follows: the pNecl-3-GFP and pNecl-3-DsRed2 vectors were digested with EcoR1 and Not1 and the resulting inserts were ligated into the plasmid pMT (Invitrogen), digested with the same enzymes.
- The resulting expression vectors were transfected into S2 Drosophila cells that were maintained in Schneider's Drosophila Medium (Gibco) supplemented with 10% FCS. The transfection was performed with Lipofactamine 2000 (Invitrogen) in 6-well culture plates: 2 µg of DNA were diluted in 50 µl of medium without serum. Lipofectamine 2000 was mixed gently before use, then diluted in 50 µl of medium, and incubated for 5 minutes at room temperature. After 5 minutes of incubation the diluted DNA was diluted in Lipofectamine 2000 (total volume =100 µl), mixed and incubated for 20 minutes at room temperature. The 100 µl of complexes were added to each well containing cells and medium. 24 hours after transfection the medium was changed and 0.5 mM CuSO4 was added for another 24 hours.
- For aggregation assays, cells to be tested were mixed after the medium change, then induced with CuSO4 and gently shaken several hours to allow for plasmid transcription, protein accumulation and aggregate formation. Hela cells were maintained in DMEM 10% FCS and transfected with Fugene (Roche) Fugene was mixed gently before use, then diluted in 100 µl of medium without serum, and incubated for 5 minutes at room temperature. After 5 minutes of incubation 2 µg of DNA were added to the diluted Fugene, mixed and incubated for 20 minutes at room temperature. The 100 µl of complexes were added to each well containing cells and medium.
- The amino acid sequence encoding human Necl-1 and -2, are presented in SEQ ID NO: 39 and SEQ ID NO: 40, respectively.
- The expression vectors of the Necl-1 and Necl-2 protein fused with GFP or with DsRed2 (respectively pMT-Necl-1-GFP/pMT-Necl-1-DsRed2 and pNecl-2-GFP which were used in the aggregation assay described in Example 6 were constructed as follows:
- The cDNA IMAGE: 5199627 (accession BC033819) of SEQ ID NO: 29 was used as a template for PCR amplification with the primers 5'-CCCCAATTGATGGGGGCCCCAGCCGC -3' of SEQ ID NO: 30 and 5'-CCCCAGATCTAAGATGAAATATTCCTTCTTGTCGTCCCC -3 of SEQ ID NO: 31. The resulting amplicon was digested with Bg12 and Mfe1 and ligated into the EcoR1 and BamH1 sites of pMT-Necl-3-GFP resulting in the pMT-Necl-1-GFP. The same digested amplicon was ligated into the EcoR1 and BamH1 sites of pMT-Necl-3-DsRed2 resulting in the pMT-Necl-1-DsRed2.
- The cDNA IMAGE: 4701395 (accession BC035930) of SEQ ID NO: 32 was used as a template for PCR amplification with the primers 5'- GGAATTCATGGCGAGTGTAGTG - 3' of SEQ ID NO: 33 and 5'- CCCCAATTGCGATGAAGTACTCTTT -3 of SEQ ID NO: 34. The resulting amplicon was digested with EcoR1 and Mfe1 and ligated into the EcoR1 site of pEGFP-N1 (BD Biosciences Clonetech) resulting in pNecl-2-GFP. The insect vector pMT-Necl-2-GFP was constructed as follows: the pNecl-2-GFP vector was digested with EcoR1 and Not1 and the resulting inserts were ligated into the plasmid pMT (Invitrogen), digested with the same enzymes.
- A bacterial expression vector pQE-Necl-3(aa169-367) was constructed as follows:
- the plasmid pNecl-3-myc obtained under Example 1 was used as a template for PCR amplification with the primers 5'-AGGTCTATATAAGC-3' of SEQ ID NO: 12 and 5'-GGGGTACCAGGGCCATTCTGGCC-3 of SEQ ID NO: 13
- the resulting amplicon was digested with Dra1 and Kpn1 and ligated into the blunted BamH1 and Kpn1 sites of the plasmid pQE-30 (Qiagen).
- The E. coli strain M15 was transformed with the bacterial expression vector pQE-Necl-3(aa169-367) and used to express the corresponding Necl-3 fragment. Bacteria were grown exponentially, IPTG was added to 1 mM, and the cells were allowed to grow for another 4 h at 37°C. Cells were then harvested and lysed. The cleared supernatants were loaded under denaturing conditions onto Ni2+-nitrilotriacetic acid agarose columns (Qiagen, 1 mL resin per 200 mL of bacteria culture), and eluted under standard conditions. The eluted proteins were then extensively dialyzed against PBS and used for immunization.
- The obtained Necl-3(aa169-367) fragment has an amino acid sequence of SEQ ID NO: 15.
- The Necl-3(aa169-367) fragment produced in E. coli as described under Example 3 and having the sequence SEQ ID NO: 15 was used to immunize two rabbits according to Harlow et al., 1988, "Antibodies: A Laboratory Manual, " Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 349 .
- This fragment was coupled to NHS activated Sepharose 4 Fast Flow (Amersham Biosciences) and used for affinity purification of the anti Necl-3 antibodies from the rabbit sera: 10 mg of protein was used for 2 mL of resin. Coupling was performed at 4 °C overnight. After the coupling, non-reacted groups on the medium were blocked with 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 for a one hour. The medium was washed after coupling with 50 mL of two buffers (0.1 M Tris-HCl buffer pH 8-9 and 0.1 M acetate buffer, 0.5 M NaCl). The antibody-containing fractions were then loaded onto a Protein A Sepharose CL-4B column (Amersham Biosciences) and the IgGs were eluted with 0.1 M glycine buffer pH 3.0).
- The antibody specificity was tested using Drosophila S2 cells transfected with either GFP alone or with Necl-1, Necl-2 (obtained as described in Example 2), Necl-3 (as described in Example 1) and Necl-4 (as described in Example 7) fused to GFP at their carboxy-termini where crude S2 lysates were separated by SDS gel electrophoresis and probed with either the anti Necl-3 (obtained as described in Example 4) or an anti GFP antibody (anti-green fluorescent protein, Roche Applied Science) serving as a loading control. The anti Necl-3 antibody reveals a single species of the expected size exclusively in cells transfected with Necl-3-GFP which proves that highly specific anti Necl-3 antibodies have been prepared which do not cross-react with other Necl/SynCAM family members, i.e. Necl-1, Necl-2, or Necl-4.
- Necl-3 mRNA expression in eighteen various rat tissues was assessed by real time PCR using a primer pair spanning two exons. The primers used for amplifying Necl-3 were SEQ ID NO: 16 (5'-TGACCATGCTCTCATAGG-3') and SEQ ID NO: 17 (5'-TGCCAGATATCGACCAAG-3'), those for GAPDH were SEQ ID NO: 18 (5'-TGATTCTACCCACGGC-3') and SEQ ID NO: 19 (5'-TGATGGGTTTCCCATTGATGA-3').
- The results confirmed northern blot data showing a Necl-3 mRNA greater than 5 Kb in various structures of the central nervous system (midbrain, cerebellum, and hippocampus), whereas it was either undetectable or poorly expressed in all other organs tested except for testis. Real time PCR analysis were performed with SYBR green (Molecular Probes) on an iCycler iQ Bio-Rad station, with quantitation, melt curve, and PCR efficiency analysis derived by the station's software as described in Pellissier et al., 2006, Anal. Biochem, 350:310-2 incorporated by reference in its entirety.
- RNA purification and cDNA synthesis were prepared as described in ( Ossipow et al., 2004, Mol Cell Neurosci. 2 7:70-83 ).
Necl-3 protein accumulation was examined by using Necl-3 antibodies obtained as described in Example 4. Western blot analysis with extracts from a series of different organs was carried out where rat tissues were heated and sonicated in Laemmli sample buffer. Proteins were separated on a 12% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes. These were probed with the appropriate antibodies and revealed by chemiluminescence (ECL, Amersham) (equal amounts of the ECL substrates were mixed and incubated with the blot for 5 min). Necl-3 protein was detected in brain structures, but also in kidney, heart and liver. Necl-3 migrates with an apparent mass of approximately 52 kDa (calculated molecular weight of 48 kDa), due to N-linked glycosylation. - The brain distribution of Necl-3 was investigated by immunohistochemistry and confocal microscopy. 2 month-old rats were anaesthetized, their brains were rapidly excised and immersed for 5 min in isopentane pre-cooled to -20°C. Brains were then embedded in OCT medium (Miles Inc., Elkhart, IN) and cryo-sectioned at 12-µm.
- Brain sections were exposed for 10 min to -20°C absolute ethanol, rinsed in PBS and incubated for 30 min in PBS containing 2% BSA. Sections were then incubated for 2 h at 37°C in PBS, 0.5% BSA, containing the affinity-purified anti-Necl-3 antibody (diluted 1:100) obtained as described under Example 4 and the appropriate mouse monoclonal antibody. After washes in PBS, Alexa Fluor highly cross-adsorbed secondary antibodies (Molecular Probes) were used to detect the mouse and rabbit primary antibodies (with Alexa 488 and Alexa 594, respectively). The brain sections were then rinsed again in PBS and stained with Hoechst. Confocal laser scanning was performed on a Leica SP2 microscope (Leica, Germany) using 20 mW blue diode (405 nm), 30 mW ArKr (488 nm), 1 mW HeNe (594 nm) lasers and a 63X plan Apo oil immersion objective. Section planes were collected in steps throughout the entire cell thickness. Images of each field were merged using the Leica Confocal software. Z-section analysis was done with the Imaris software (Bitplane AG, Switzerland) using the "Section" function with the stacks series of confocal acquisitions.
- Intense labeling was observed in structures resembling axon bundles in several brain areas including cerebellar white matter and ventral reticular nucleus. The axonal localization of Necl-3 was confirmed by a near-perfect overlap with the axonal neurofilament marker.
- Monoclonal antibodies directed against the MBP (Calbiochem) were used in combination with anti Necl-3 antibodies obtained as described under Example 4 to confirm that Necl-3 is found in the axoplasm of myelinated axons and that Necl-3 immunoreactivity is stronger in the myelinated areas.
- S2 cells do not aggregate spontaneously and the ability of Necl-3 to induce their aggregation was assayed. S2 cells were transfected separately with plasmids expressing GFP (the insect cell expression vector pMT-GFP was constructed as follows: pEGFP-N1 (BD Biosciences Clonetech) was digested with EcoR1 and Not1, and the resulting insert was ligated into the plasmid pMT (Invitrogen), digested with the same enzymes) or Necl-3-GFP obtained under Example 1 under the control of the drosophila inducible metallothionein promoter. After transfection, the cells were washed free from plasmid, and mixed together. They were then induced with CuSO4, gently shaken to favor aggregation and visualized without further fixation. Unlike cells transfected with GFP, those transfected with Necl-3-GFP formed large aggregates. Non-transfected cells serving as internal control did not aggregate.
- S2 cells were separately transfected with plasmids coding for Necl-3-GFP (Example 1), Necl-1-DsRed2 (Example 2), and Necl-4-DsRed2 (Example 7). After transfection, they were mixed together in different combinations and induced to express their transfected Necl. After gentle shaking, aggregates formed between Necl-3-GFP and Necl-1-DsRed2 or Necl-4-DsRed2 were observed by fluorescence microscopy. A similar S2 cells aggregation assay using a myc-tagged version of Necl-3 (pNecl-3-myc) along with Necl-2-GFP. Using a rhodamine-conjugated anti myc secondary antibody, aggregates of red and green cells were again observed.
- Thus, Necl-3 unambiguously interacts with itself, Necl-1, Necl-2, and Necl-4.
- An Fc-Necl-3 fusion protein between the Fc domain of a human IgG1 of SEQ ID NO: 35 and the extra-cellular domain of Necl-3 (aa 1-365) of SEQ ID NO: 2 was prepared as follows: First, a mammalian expression vector pFc-Necl-3 was constructed by using the plasmid pNecl-3-myc (Example 1) as a template for PCR amplification with the primers 5'-AGGTCTATATAAGC-3' of SEQ ID NO: 12 and 5'-GGGGTACCAGGGCCATTCTGGCC-3 of SEQ ID NO: 13. The resulting amplicon was digested with Nhe1 and Kpn1 and ligated into the plasmid pIgplus (R&D Systems, Abingdon, United Kingdom) digested with the same enzymes, resulting in pFc-Necl-3. An insect cell expression vector pMT-Fc-Necl-3 was then constructed wherein the pFc-Necl-3 vector obtained above was digested with Nhe1 and Apa1 and the resulting insert was ligated into the plasmid pMT (Invitrogen) digested with Spe1 and Apa1 resulting in pMT-Fc-Necl-3.
- The obtained insect cell expression vector pMT-Fc-Necl-3 was then co-transfected in S2 cells along with the plasmid pCoBlast that carries a blasticidin S resistance gene (Invitrogen). Stable clones were selected with blasticidin S at 25 µg/mL. Of these, high expressers of the Fc-Necl-3 fusion were selected. One such clone was expanded and grown in a 150 mL flat bottom flask and induced with 0.5 mM CuSO4. After 4 days, the conditioned culture medium was loaded onto a Protein A Sepharose CL-4B column (Amersham Biosciences). The Fc-Necl-3 fusion protein which was bond to the resin directly from the culture medium was recovered by washing the column (10 volumes of Tris-HCl: 50 mM, pH 7.4) and eluting (0.1 M glycine buffer pH 3.0), and was then characterised by western blotting.
- This fusion protein or a control full-length human IgGs (Sigma) were adsorbed on protein A resin. 100µl of lysates of mock-transfected and pMT-Necl-2-GFP transfected S2 cells (Example 2) were pre-cleared for 30 min at 4°C with 25 µl of Protein A Sepharose CL-4B resin and used as the inputs for the following pull-down experiments:
- After pre-clearing, the lysates were rocked 45 min at 4°C with the Fc-Necl-3 fusion protein immobilized on Protein A Sepharose CL-4B resin. After incubation, the proteins retained on the beads were washed 3 times with 500 µl RIPA buffer, and boiled in 1X Laemmli sample buffer for western blot analysis. After incubation and washings, the proteins retained on the beads were analyzed by western blot. Control IgG beads were unable to bring down Necl-2-GFP, whereas the Fc-Necl-3 beads efficiently did so. This shows that Necl-3 is able to make heterophilic interactions with Necl-2, and the fact that human IgGs beads do not interact with Necl-2 confirms that even though the Necl proteins are capable of multiple heterophilic interactions, they retain specificity.
- Necl-4 amplification was performed by PCR reaction with Pfu Turbo polymerase (Stratagene): 1X cloned Pfu buffer, 200 µM each dNTP, 0.1-0.5 µM each primer, 2.5 units cloned enzyme, 1-100 ng plasmid template DNA, or 100-250 ng genomic template DNA). Insect expression vectors pMT-Necl-4-GFP and pMT-Necl-4-DsRed2 were constructed as follows:
- the modified cDNA clone IMAGE 4375136 (accession BC068457) mutated in position 563 wherein A is mutated in G as shown on SEQ ID NO: 20 was used as a template for PCR amplification with the primers 5'-CCCCAATTGATGGGCCGGGCCCGG-3' of SEQ ID NO: 21 and 5'-CCCCAGATCTAAGATGAAGAATTCCTCTTTCCTCTTGT-3 of SEQ ID NO: 22.
- the resulting amplicon was digested with Mfe1 and Bg12 and ligated into either a pMT-GFP or a pMT-DsRed2 digested by EcoR1 and BamH1.
- A mammalian expression vector pNecl-4-GFP was constructed as follows:
- pMT-Necl-4-GFP obtained above was digested with Xba1 and the fragment Necl-4-GFP was ligated into the pcDNA3.1/myc-His (Invitrogen) digested with the same enzyme.
- A bacterial expression vector pQE-Necl-4(aa126-322) was constructed as follows:
- the plasmid pNecl-4-GFP above was used as a template for PCR amplification with the primers 5'-CCCGCATGCAATCCTGTGGTGGAGGTC-3' of SEQ ID NO: 23 and 5'-CCCCTGCAGGGGAACCGACGTCTGA-3 of SEQ ID NO: 24.
- the resulting amplicon was digested with Sph1 and Pst1 and ligated into the pQE-30 (Qiagen), digested with the same enzymes.
- The insect vectors were transfected into S2 Drosophila cells under the same conditions as described for the transfection conditions described in Example 1.
- A M15 E. coli strain was transformed with the bacterial expression vector pQE-Necl-4(aa126-322) obtained above and used to express the corresponding Necl-4 fragment. The bacteria were grown exponentially, 1 mM IPTG was added, and the cells were allowed to grow for another 4 h at 37°C. Cells were then harvested and lysed. The cleared supernatants were loaded under denaturing conditions onto Ni2+-nitrilotriacetic acid agarose columns (Qiagen), i.e. 1 mL resin per 200 mL of bacteria culture, and eluted under standard conditions. The eluted proteins were then extensively dialyzed against PBS and used for immunization. The obtained Necl-4(aa126-322) fragment has an amino sequence of SEQ ID NO: 26.
- The Necl-4(aa126-322) fragment produced in E. coli as described under Example 7 and having the amino acid sequence of SEQ ID NO: 26 was used by to immunize two rabbits on the same manner as described under Example 4.
- Necl-4 expression was examined in various tissues by real-time PCR in the way as described under Example 5 and wherein the primers used for amplifying Necl-4 were 5'-GAGGGCGCTCTACGTACTTG-3' of SEQ ID NO: 27 and 5'-AACCGACGTCTGAGCCTCTA-3' of SEQ ID NO: 28, those for GAPDH were of SEQ ID NO: 18 and SEQ ID NO: 19.
- The Necl-4 expression was shown to be the highest in brain and spinal cord.
- The ability of Necl-4 to form homophilic interaction was demonstrated as described under Example 6 wherein the S2 cells were transfected with the plasmid expressing Necl-4-GFP obtained under Example 7.
- The ability of Necl-4 to form heterophilic interaction was demonstrated as described under Example 6 wherein the S2 cells were separately transfected with the plasmid expressing Necl-4-GFP obtained under Example 7, the plasmid coding for Necl-3-GFP obtained under Example 1, the plasmids coding for Necl-4-DsRed2 obtained under Example 7, Necl-1-GFP and Necl-2-GFP obtained under Example 2.
- Necl-4 showed to unambiguously interact with itself, Necl-1, Necl-2, and Necl-3.
- The following fusion proteins for modulating the Necl-3 and/or Necl-4 induced cell aggregation are prepared as described in Example 6.
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the mouse Fc domain of an IgG2a of SEQ ID NO: 37;
- The fusion protein formed by the human Necl-3 extracellular domain (aa 1-365) of SEQ ID NO: 4 fused to the mouse Fc domain of an IgG3 of SEQ ID NO: 38;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the mouse Fc domain of an IgG2a of SEQ ID NO: 37; and
- The fusion protein formed by the human Necl-4 extracellular domain (aa 1-323) of SEQ ID NO: 8 fused to the mouse Fc domain of an IgG3 of SEQ ID NO: 38.
- The efficiency of the Necl-3 antagonists or Necl-4 antagonists and in particular of fusion proteins according to the invention in the treatment of CNS disorders may be assayed in different models such as Experimental Autoimmune Encephalomyelitis (EAE) model such as described in Loo et al., 2006, Nature Immunology 7(9): 954-961 . In particular, Necl-3 antagonists or Necl-4 antagonists according to the invention may be subjected to i.v. injection with various doses and dosing regimens in mice primed to develop (EAE). Specifically, a MOG peptide (SEQ ID NO: 41) (Sigma-Aldrich Co.) is injected into mice of 10-15 weeks of age by subcutaneous injection of 200 µg peptide in 200 µl sterile PBS emulsified with an equal volume of complete Freund's adjuvant (Sigma) containing 5 mg/mL of Mycobacterium tuberculosis H37Ra (BD). The effect of Necl-3 antagonists or Necl-4 antagonists according to the invention on the disease course are assigned with scores on a scale of 0-6, with 0.5 points for immediate clinical findings, as follows: 0, normal; 1, weakness of the tail; 2, complete loss of tail tonicity; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, forelimb paralysis or moribund; and 6, death.
Claims (22)
- An isolated polypeptide having at least 80% identity or homology with a sequence of amino acids selected from the group consisting of:(a) amino acids 1 to 435 of SEQ ID NO: 2;(b) amino acids 1 to 365 SEQ ID NO: 4;(c) amino acids 1 to 388 of SEQ ID NO: 6;(d) amino acids 1 to 323 of SEQ ID NO: 8;(e) amino acids 1 to 198 of SEQ ID NO: 15; and(f) amino acids 1 to 198 of SEQ ID NO: 26.
- An isolated polypeptide according to claim 1 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 15 and SEQ ID NO: 26.
- An isolated nucleic acid consisting of a nucleotide sequence encoding a polypeptide having at least 80% identity or homology with a sequence of amino acids selected from the group consisting of:(a) amino acids 1 to 435 of SEQ ID NO: 2;(b) amino acids 1 to 365 SEQ ID NO: 4;(c) amino acids 1 to 388 of SEQ ID NO: 6;(d) amino acids 1 to 323 of SEQ ID NO: 8;(e) amino acids 1 to 198 of SEQ ID NO: 15; and(f) amino acids 1 to 198 of SEQ ID NO: 26.
- A fusion protein comprising the following sequences:- an effector region having a sequence of amino acids according to claims 1 or 2;- an immunoglobulin constant region.
- A fusion protein according to claim 4 wherein the effector region is a protein with an amino acid sequence of SEQ ID NO: 4.
- A fusion protein according to claim 4 wherein the effector region is a protein with an amino acid sequence of SEQ ID NO: 8.
- A fusion protein according to any of claims 4 to 6 wherein the immunoglobulin constant region is the Fc domain of an IgG.
- A fusion protein according to any of claims 4 to 7 selected from the following group:A fusion protein formed by SEQ ID NO: 4 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35;A fusion protein formed by SEQ ID NO: 4 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36;A fusion protein formed by SEQ ID NO: 8 fused to the human Fc domain of an IgG1 of SEQ ID NO: 35; andA fusion protein formed by SEQ ID NO: 8 fused to the human Fc domain of an IgG4 of SEQ ID NO: 36.
- A fusion protein according to any one of claims 4 to 8 for use as a medicament.
- A pharmaceutical composition comprising fusion protein according to any one of claims 4 to 8 and a physiologically acceptable carrier, diluent or excipient.
- Use of a Necl-3 antagonist or a Necl-4 antagonist for the manufacture of a medicament for the treatment of CNS disorders comprising neuroinflammatory and neurodegenerative conditions or demyelinating disorders or diseases.
- A use according to claim 11 wherein the Necl-3 antagonist or the Necl-4 antagonist is a fusion protein according to any one of claims 4 to 8.
- A use according to claims 11 or 12 wherein the CNS disorder is multiple sclerosis.
- A use according to any one of claims 11 to 13 wherein the Necl-3 antagonist or the Necl-4 antagonist is to be administered in combination with a co-agent useful in the treatment of CNS disorders.
- An isolated DNA sequence that encodes a fusion protein according to any one of claims 4 to 8.
- A recombinant expression vector comprising a nucleic acid molecule according to claim 3, wherein the vector optionally comprises an expression control sequence, allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide, operably linked to the nucleic acid molecule.
- A host cell transfected or transformed with a recombinant expression vector according to claim 16 or a nucleic acid according to claim 3.
- A process for producing cells capable of expressing a polypeptide according to claims 1 or 2 or a fusion protein according to any one of claims 4 to 8 comprising genetically engineering cells with a vector according to claim 16 or a nucleic acid according to claim 3.
- A kit comprising at least one fusion protein according to any one of claims 4 to 8.
- A process for producing a fusion protein according to any one of claims 4 to 8 comprising culturing a host cell transformed with an expression vector according to claim 16 under conditions that promotes expression of said fusion protein and recovering said fusion protein.
- A method of antagonizing the aggregation of Necl-3 and/or of Necl-4 with itself of with at least one protein of the Necl family, comprising exposing cells that express Necl-3 or Necl-4, to a fusion protein according to any one of claims 4 to 8, such that the aggregation of said Necl-3 or Necl-4 with itself or with at least one protein of the Necl family is antagonized.
- A method for screening for a Necl-3 and/or a Necl-4 antagonist comprising the following steps:(i) Combining cells that do not aggregate spontaneously in presence of a Necl-3 and/or a Necl-4 protein with at least one peptide according to claim 1 or 2, in presence/absence of a compound to be screened;(ii) Determining the ability of the compound to inhibit the Necl-3 and/or Necl-4 induced cell aggregation.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06123480A EP1918299A1 (en) | 2006-11-03 | 2006-11-03 | Modulators of cell adhesion molecules and use thereof |
PCT/EP2007/061856 WO2008053049A1 (en) | 2006-11-03 | 2007-11-05 | Modulators of cell adhesion molecules and use thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP06123480A EP1918299A1 (en) | 2006-11-03 | 2006-11-03 | Modulators of cell adhesion molecules and use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1918299A1 true EP1918299A1 (en) | 2008-05-07 |
Family
ID=37898581
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP06123480A Withdrawn EP1918299A1 (en) | 2006-11-03 | 2006-11-03 | Modulators of cell adhesion molecules and use thereof |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP1918299A1 (en) |
WO (1) | WO2008053049A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2322656A1 (en) * | 2009-11-17 | 2011-05-18 | Centre National de la Recherche Scientifique (C.N.R.S) | Methods for diagnosing skin diseases |
US8420084B2 (en) | 2009-03-05 | 2013-04-16 | Medarex, Inc. | Fully human antibodies specific to CADM1 |
US9903872B2 (en) | 2006-08-29 | 2018-02-27 | Oxford Biotherapeutics, Ltd. | Identification of protein associated with hepatocellular carcinoma, gliobastoma and lung cancer |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100150924A1 (en) * | 2007-05-22 | 2010-06-17 | Elior Peles | Regulation of myelination by nectin-like (necl) molecules |
WO2011005523A2 (en) * | 2009-06-23 | 2011-01-13 | The Brigham And Women's Hospital, Inc. | Galectin-immunoglobulin chimeric molecules |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000032633A1 (en) * | 1998-12-02 | 2000-06-08 | Icos Corporation | Novel adhesion molecule and methods of use |
WO2002066643A2 (en) * | 2000-11-13 | 2002-08-29 | Curagen Corporation | Proteins, polynucleotides encoding them and methods of using the same |
US20030109027A1 (en) * | 2001-10-11 | 2003-06-12 | President, National Cancer Center | TSLL2 gene |
US20030199103A1 (en) * | 1999-06-03 | 2003-10-23 | Curagen Corporation | Novel amino acid sequences for human epidermal growth factor-like polypeptides |
-
2006
- 2006-11-03 EP EP06123480A patent/EP1918299A1/en not_active Withdrawn
-
2007
- 2007-11-05 WO PCT/EP2007/061856 patent/WO2008053049A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000032633A1 (en) * | 1998-12-02 | 2000-06-08 | Icos Corporation | Novel adhesion molecule and methods of use |
US20030199103A1 (en) * | 1999-06-03 | 2003-10-23 | Curagen Corporation | Novel amino acid sequences for human epidermal growth factor-like polypeptides |
WO2002066643A2 (en) * | 2000-11-13 | 2002-08-29 | Curagen Corporation | Proteins, polynucleotides encoding them and methods of using the same |
US20030109027A1 (en) * | 2001-10-11 | 2003-06-12 | President, National Cancer Center | TSLL2 gene |
Non-Patent Citations (11)
Title |
---|
ARASE NORIKO ET AL: "Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8(+) T cells", INTERNATIONAL IMMUNOLOGY, vol. 17, no. 9, September 2005 (2005-09-01), pages 1227 - 1237, XP002428924, ISSN: 0953-8178 * |
BIEDERER ET AL: "Bioinformatic characterization of the SynCAM family of immunoglobulin-like domain-containing adhesion molecules", GENOMICS, ACADEMIC PRESS, SAN DIEGO, US, vol. 87, no. 1, January 2006 (2006-01-01), pages 139 - 150, XP005226285, ISSN: 0888-7543 * |
CHANG GUIMIN ET AL: "Mouse Necl-3 promoter methylation in TRAMP cells", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 47, April 2006 (2006-04-01), & 97TH ANNUAL MEETING OF THE AMERICAN-ASSOCIATION-FOR-CANCER-RESEARCH (AACR); WASHINGTON, DC, USA; APRIL 01 -05, 2006, pages 376, XP001249379, ISSN: 0197-016X * |
CHANG GUIMIN ET AL: "Perturbation of endogenous necl-3 expression induces tumorigenic phenotypes of polarized MDCK cells.", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 47, April 2006 (2006-04-01), & 97TH ANNUAL MEETING OF THE AMERICAN-ASSOCIATION-FOR-CANCER-RESEARCH (AACR); WASHINGTON, DC, USA; APRIL 01 -05, 2006, pages 640, XP001249380, ISSN: 0197-016X * |
DONG XIUHUA ET AL: "Crystal structure of the V domain of human Nectin-like molecule-1/Syncam3/Tsll1/Igsf4b, a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule.", THE JOURNAL OF BIOLOGICAL CHEMISTRY 14 APR 2006, vol. 281, no. 15, 14 April 2006 (2006-04-14), pages 10610 - 10617, XP002428927, ISSN: 0021-9258 * |
FUKAMI T ET AL: "Isolation of the mouse Tsll1 and Tsll2 genes, orthologues of the human TSLC1-like genes 1 and 2 (TSLL1 and TSLL2)", GENE: AN INTERNATIONAL JOURNAL ON GENES AND GENOMES, ELSEVIER, AMSTERDAM, NL, vol. 323, 24 December 2003 (2003-12-24), pages 11 - 18, XP004477026, ISSN: 0378-1119 * |
FURUNO TADAHIDE ET AL: "The spermatogenic Ig superfamily/synaptic cell adhesion molecule mast-cell adhesion molecule promotes interaction with nerves", JOURNAL OF IMMUNOLOGY, vol. 174, no. 11, June 2005 (2005-06-01), pages 6934 - 6942, XP002428925, ISSN: 0022-1767 * |
ITO AKIHIKO ET AL: "Direct interaction between nerves and mast cells mediated by the SgIGSF/SynCAM adhesion molecule.", JOURNAL OF PHARMACOLOGICAL SCIENCES SEP 2006, vol. 102, no. 1, September 2006 (2006-09-01), pages 1 - 5, XP002428926, ISSN: 1347-8613 * |
KOMA YU-ICHIRO ET AL: "Cloning of a soluble isoform of the SgIGSF adhesion molecule that binds the extracellular domain of the membrane-bound isoform", ONCOGENE, vol. 23, no. 33, 22 July 2004 (2004-07-22), pages 5687 - 5692, XP002428923, ISSN: 0950-9232 * |
OGITA HISAKAZU ET AL: "Nectins and nectin-like molecules: Roles in cell adhesion, polarization, movement, and proliferation", IUBMB LIFE, vol. 58, no. 5-6, May 2006 (2006-05-01), pages 334 - 343, XP009081927, ISSN: 1521-6543 * |
WILLIAMS Y N ET AL: "Cell adhesion and prostate tumor-suppressor activity of TSLL2/IGSF4C, an immunoglobulin superfamily molecule homologous to TSLC1/IGSF4", ONCOGENE, vol. 25, no. 10, March 2006 (2006-03-01), pages 1446 - 1453, XP002440672, ISSN: 0950-9232 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9903872B2 (en) | 2006-08-29 | 2018-02-27 | Oxford Biotherapeutics, Ltd. | Identification of protein associated with hepatocellular carcinoma, gliobastoma and lung cancer |
US8420084B2 (en) | 2009-03-05 | 2013-04-16 | Medarex, Inc. | Fully human antibodies specific to CADM1 |
EP2322656A1 (en) * | 2009-11-17 | 2011-05-18 | Centre National de la Recherche Scientifique (C.N.R.S) | Methods for diagnosing skin diseases |
WO2011061184A1 (en) * | 2009-11-17 | 2011-05-26 | Centre National De La Recherche Scientifique (C.N.R.S) | Methods for diagnosing skin diseases |
Also Published As
Publication number | Publication date |
---|---|
WO2008053049A1 (en) | 2008-05-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US9777058B2 (en) | Methods of treating a tauopathy | |
US9044428B2 (en) | Neuritogenic peptides | |
KR100767146B1 (en) | Humanized Antibodies That Sequester Amyloid Beta Peptide | |
EP4001305A9 (en) | Anti-tau antibody and use of same | |
JP6797810B2 (en) | Anti-CD40L Antibodies and Methods for Treating CD40L-Related Diseases or Disorders | |
US8008259B2 (en) | Neurotrophin-derived peptide sequences | |
EP2313432B1 (en) | Use of cd31 peptides in the treatment of thrombotic and autoimmune disorders | |
CA3173775A1 (en) | Methods of treating a tauopathy | |
JP2006109840A (en) | Protocadherin, its immune body and use | |
EP3292144B1 (en) | Use of cd24 for lowering low-density lipoprotein cholesterol levels | |
EP1918299A1 (en) | Modulators of cell adhesion molecules and use thereof | |
US20220213163A1 (en) | Il-2 chimeric proteins for immunosuppression | |
US20220098278A1 (en) | Methods of Use of Soluble CD24 for Neuroprotection and Remyelination | |
JP2018529729A (en) | Treatment of bile acid disorders | |
EP3353205A1 (en) | Anti-mullerian hormone (amh) neutralizing antibodies and uses thereof | |
US20100040623A1 (en) | Neuritogenic and neuronal survival promoting peptides derived from the family of s-100 proteins | |
JP2022513338A (en) | Anti-CD79 antibody and its use | |
EP3072527B1 (en) | Immunosuppressant | |
WO2012101125A1 (en) | Specific antibodies against human cxcl4 and uses thereof | |
US20220000974A1 (en) | Targeting CD24-Siglec Interactions for Treating Subjects with Prediabetes or Diabetes | |
EP4082574A1 (en) | Agent for preventing or treating acute-phase neuromyelitis optica | |
WO2023154953A1 (en) | Gdf15 polypeptides for treating and preventing autoimmune diseases |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR |
|
AX | Request for extension of the european patent |
Extension state: AL BA HR MK RS |
|
AKX | Designation fees paid | ||
REG | Reference to a national code |
Ref country code: DE Ref legal event code: 8566 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20081110 |